Retroviral vectors

ABSTRACT

A retroviral vector derived from a non-primate lentivirus genome comprising a deleted gag gene wherein the deletion in gag removes one or more nucleotides downstream of nucleotide 350 of the gag coding sequence.

[0001] This invention relates to a retroviral vector. In particular, butnot exclusively, it relates to retroviral vectors capable oftransferring genetic material to non-dividing or slowly-dividing cellsderived from non-primate lentiviruses.

[0002] There has been considerable interest, for some time, in thedevelopment of retroviral vector systems based on lentiviruses, a smallsubgroup of the retroviruses. This interest arises firstly from thenotion of using HIV-based vectors to target anti-HIV therapeutic genesto HIV susceptible cells and secondly from the prediction that, becauselentiviruses are able to infect non-dividing cells (Lewis & Emerman 1993J. Virol. 68, 510), vector systems based on these viruses would be ableto transduce non-dividing cells (e.g. Vile & Russel 1995 Brit. Med.Bull. 51, 12). Vector systems based on HIV have been produced(Buchschacher & Panganiban 1992 J. Virol. 66, 2731) and they have beenused to transduce CD4+cells and, as anticipated, non-diving cells(Naldini et al, 1996 Science 272, 263). In addition lentiviral vectorsenable very stable long-term expression of the gene of interest. Thishas been shown to be at least three months for transduced rat neuronalcells. The MLV based vectors were only able to express the gene ofinterest for six weeks.

[0003] HIV-based vectors produced to date result in an integratedprovirus in the transduced cell that has HIV LTRs at its ends. Thislimits the use of these vectors as the LTRs have to be used asexpression signals for any inserted gene unless an internal promoter isused. The use of internal promoters has significant disadvantages. Theunpredictable outcome of placing additional promoters within theretroviral LTR transcription unit is well documented (Bowtell et al,1988 J. Virol. 62, 2464; Correll et al, 1994 Blood 84, 1812; Emerman andTemin 1984 Cell 39, 459; Ghattas et al, 1991 Mol. Cell. Biol. 11, 5848;Hantzopoulos et al, 1989 PNAS 86, 3519; Hatzoglou et al, 1991 J. Biol.Chem 266, 8416; Hatzoglou et al, 1988 J. Biol. Chem 263, 17798; Li etal, 1992 Hum. Gen. Ther. 3, 381; McLaichlin et al, 1993 Virol. 195, 1;Overell et al, 1988 Mol. Cell Biol. 8, 1803; Scharfman et al, 1991 PNAS88, 4626; Vile et al, 1994 Gene Ther 1, 307; Xu et al, 1989 Virol. 171,331; Yee et al, 1987 PNAS 84, 5197). The factors involved appear toinclude the relative position and orientation of the two promoters, thenature of the promoters and the expressed genes and any selectionprocedures that may be adopted. The presence of internal promoters canaffect both the transduction titers attainable from a packaging cellline and the stability of the integrated vector.

[0004] HIV and other lentiviral LTRs have virus-specific requirementsfor gene expression. For example, the HIV LTR is not active in theabsence of the viral Tat protein (Cullen 1995 AIDS 9, S19). It isdesirable, therefore, to modify the LTRs in such a way as to change therequirements for gene expression. In particular tissue specific geneexpression signals may be required for some gene therapy applications.

[0005] HIV vectors have a number of significant disadvantages which maylimit their therapeutic application to certain diseases. HIV-1 has thedisadvantage of being a human pathogen carrying potentially oncogenicproteins and sequences. There is the risk that introduction of vectorparticles produced in packaging cells which express HIV gag-pol willintroduce these proteins into the patient leading to seroconversion. Forthese reasons, there is a need to develop lentiviral-based vectors whichdo not introduce HIV proteins into patients.

[0006] We have now found it possible to provide an improved lentiviralvector which overcomes the limitations of HIV-based vectors. It isimportant in the development of any retroviral vector system to removesequences from the retroviral genome which may inhibit the capacity ofthe vector to transfer heterologous genes or which may transferdisadavantageous protein coding sequences to the target cell.Retroviruses are limited in the length of RNA sequences which can bepackaged efficiently and so the existence of long regions of theretroviral genome severely limits the coding capacity of the vector forheterologous coding RNA.

[0007] We have also found it possible to provide a lentiviral vectorbased on a non-primate lentivirus which has a high coding capacity forheterologous coding sequences and which has a reduced capacity totransfer retroviral components to the target cell.

[0008] It has surprisingly been found that the amount of vector genomicsequence required from a non-primate lentivirus to produce an efficientcloning vector is substantially less than has been described for anHIV-based vector.

[0009] The sequence requirements for packaging HIV vector genomes arecomplex. The HIV-1 packaging signal encompasses the splice donor siteand contains a portion of the 5′-untranslated region of the gag genewhich has a putative secondary structure containing 4 short stem-loops.Additional sequences elsewhere in the genome are also known to beimportant for efficient encapsidation of HV. For example the first 350bps of the gag protein coding sequence may contribute to efficientpackaging and ill defined regions of env are also implicated. For theconstruction of HIV-vectors capable of expressing heterologous genes, apackaging signal extending to 350 bps of the gag protein-coding regionhas been used. We have surprisingly found that the structure of thepackaging signal in non-primate lentiviruses is entirely different fromthat of HIV. Instead of a short sequence of 4 stem loops followed by anill defined region of gag and env sequences, we have discovered that ashorter region of the gag gene suffices for efficient packaging. Indeeddeletion of larger regions of the gag gene in EIAV vectors isadvantageous and leads to higher titre viral vector being produced. Thisinformation can be used to provide improved vectors constructed fromnon-primate lentivirus sequences which have high titre and advantageousfeatures compared to HV vectors.

[0010] In a first aspect of the invention, there is provided aretroviral vector genome containing a deleted gag gene from anon-primate lentivirus wherein the deletion in gag removes one or morenucleotides downstream of nucleotide 350 of the gag coding sequence.Preferably the deletion extends from nucleotide 350 to at least theC-terminus of the gagpol coding region. More preferably the deletionadditionally removes nucleotide 300 of the gag coding region and mostpreferably the deletion retains only the first 150 nucleotides of thegag coding region. However even larger deletions of gag can also beused, for example the gag coding region contains the first 109nucleotides of the gag coding region. It may also be possible for thegag coding region to contain only the first 2 nucleotides of the gagcoding region.

[0011] Additional features of the lentiviral genome are included in thevector genome which are necessary for transduction of the target cell;replication; reverse transcription and integration. These are, at least,a portion of an LTR containing sequence from the R-region and U5 region,sequences from the 3′ LTR which contain a polypurine tract (PT) and a 3′LTR from the non-primate lentivirus or a hybrid LTR containing sequencesfrom the non-primate lentivirus and other elements. Optionally, theretroviral genome may contain accessory genes derived from a retrovirus,such as, but not limited to, a rev gene, a tat gene, a vif gene, a nefgene, a vpr gene or an S2 gene. Additional components may be added suchas introns, splice-donor sites, a rev responsive element (RRE), cloningsites and selectable marker genes.

[0012] Moreover, we have now surprisingly demonstrated that anon-primate lentivirus minimal vector system can be constructed whichrequires neither S2, Tat, env nor dUTPase for either vector productionor for transduction of dividing and non-dividing cells.

[0013] Thus according to another aspect the non-primate lentivirusgenome from which the vector is derived lacks one or more accessorygenes.

[0014] The deletion of accessory genes is highly advantageous. Firstly,it permits vectors to be produced without the genes normally associatedwith disease in lentiviral (e.g. HIV) infections. In particular, tat andenv are associated with disease. Secondly, the deletion of accessorygenes permits the vector to package more heterologous DNA. Thirdly,genes whose function is unknown, such as dUTPase and S2, may be omitted,thus reducing the risk of causing undesired effects.

[0015] In addition, we have shown that the leader sequence of thenon-primate lentivirus genome is essential for high protein expressionof gag and gagpol.

[0016] Therefore in a further aspect the non-primate lentivirus genomefrom which the vector is derived lacks the tat gene but includes theleader sequence between the end of the 5′ LTR and the ATG of gag.

[0017] These data further define a minimal essential set of functionalcomponents for an optimal lentiviral vector. A vector is provided withmaximal genetic capacity and high titre, but without accessory genesthat are either of unknown function (S2, UTPase), and therefore maypresent risk, or are analogues of HIV proteins that may be associatedwith AIDS (tat, env).

[0018] It will be appreciated that the present invention provides aretroviral vector derived from a non-primate lentivirus genome (1)comprising a deleted gag gene wherein the deletion in gag removes one ormore nucleotides downstream of nucleotide 350 of the gag codingsequence; (2) wherein one or more accessory genes are absent from thenon-primate lentivirus genome; (3) wherein the non-primate lentivirusgenome lacks the tat gene but includes the leader sequence between theend of the 5ζ LTR and the ATG of gag; and combinations of (1), (2) and(3). In a preferred embodiment the retroviral vector comprises all offeatures (1) and (2) and (3).

[0019] A “non-primate” vector, as used herein, refers to a vectorderived from a virus which does not primarily infect primates,especially humans. Thus, non-primate virus vectors include vectors whichinfect non-primate mammals, such as dogs, sheep and horses, reptiles,birds and insects.

[0020] A lentiviral or lentivirus vector, as used herein, is a vectorwhich comprises at least one component part derived from a lentivirus.Preferably, that component part is involved in the biological mechanismsby which the vector infects cells, expresses genes or is replicated.

[0021] The non-primate lentivirus may be any member of the family oflentiviridae which does not naturally infect a primate and may include afeline immunodeficiency virus (F1v), a bovine immunodeficiency virus(BIV), a caprine arthritis encephalitis virus (CAEV), a Maedi visnavirus (MVV) or an equine infectious anaemia virus (EIAV). Preferably thelentivirus is an EIAV. Equine infectious anaemia virus infects allequidae resulting in plasma viremia and thrombocytopenia (Clabough, etal. 1991. J Virol. 65:6242-51). Virus replication is thought to becontrolled by the process of maturation of monocytes into macrophages.

[0022] EIAV has the simplest genomic structure of the lentiviruses. Inaddition to the gag, pol and env genes EIAV encodes three other genes:tat, rev, and S2. Tat acts as a transcriptional activator of the viralLTR (Derse and Newboldl993 Virology. 194:530-6; Maury, et al 1994Virology. 200:632-42.) and Rev regulates and coordinates the expressionof viral genes through rev-response elements (RRE) (Martarano et al 1994J Virol. 68:3102-11.). The mechanisms of action of these two proteinsare thought to be broadly similar to the analogous mechanisms in theprimate viruses (Martano et al ibid). The function of S2 is unknown. Inaddition, an EIAV protein, Ttm, has been identified that is encoded bythe first exon of tat spliced to the env coding sequence at the start ofthe transmembrane protein.

[0023] In addition to protease, reverse transcriptase and integrasenon-primate lentiviruses contain a fourth pol gene product which codesfor a dUTPase. This may play a role in the ability of these lentivirusesto infect certain non-dividing cell types.

[0024] The viral RNA in the first aspect of the invention is transcribedfrom a promoter, which may be of viral or non-viral origin, but which iscapable of directing expression in a eukaryotic cell such as a mammaliancell. Optionally an enhancer is added, either upstream of the promoteror downstream. The RNA transcript is terminated at a polyadenylationsite which may be the one provided in the lentiviral 3ζ LTR or adifferent polyadenylation signal.

[0025] Thus the present invention provides a DNA transcription unitcomprising a promoter and optionally an enhancer capable of directingexpression of a retroviral vector genome.

[0026] Transcription units as described herein comprise regions ofnucleic acid containing sequences capable of being transcribed. Thus,sequences encoding MRNA, tRNA and rRNA are included within thisdefinition. The sequences may be in the sense or antisense orientationwith respect to the promoter. Antisense constructs can be used toinhibit the expression of a gene in a cell according to well-knowntechniques. Nucleic acids may be, for example, ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or analogues thereof. Sequences encodingmRNA will optionally include some or all of 5′ and/or 3′ transcribed butuntranslated flanking sequences naturally, or otherwise, associated withthe translated coding sequence. It may optionally further include theassociated transcriptional control sequences normally associated withthe transcribed sequences, for example transcriptional stop signals,polyadenylation sites and downstream enhancer elements. Nucleic acidsmay comprise cDNA or genomic DNA (which may contain introns).

[0027] The term “promoter” is used in the normal sense of the art, e.g.an RNA polymerase binding site in the Jacob-Monod theory of geneexpression.

[0028] The term “enhancer” includes a DNA sequence which binds to otherprotein components of the transcription initiation complex and thusfacilitates the initiation of transcription directed by its associatedpromoter.

[0029] The promoter and enhancer of the transcription units encoding thefirst viral vector component are preferably strongly active, or capableof being strongly induced, in the producer cell under conditions forproduction of the retroviral vector of the present invention and/or inprimary target cells under conditions for production of the secondaryviral vector. The promoter and enhancer of the transcription unitsencoding the second viral vector component are preferably stronglyactive, or capable of being strongly induced, in the target cells. Thepromoter and/or enhancer may be constitutively efficient, or may betissue or temporally restricted in their activity. Examples of suitabletissue restricted promoters/enhancers are those which are highly activein tumour cells such as a promoter/enhancer from a MUC1 gene, a CEA geneor a 5T4 antigen gene. Examples of temporally restrictedpromoters/enhancers are those which are responsive to ischaemia and/orhypoxia, such as hypoxia response elements or the promoter/enhancer of agrp78 or a grp94 gene. One preferred promoter-enhancer combination is ahuman cytomegalovirus (hCMV) major immediate early (MIE)promoter/enhancer combination.

[0030] The LTRs may be altered in, for example, U3 (such as to obtainstrong constitutive expression, inducible expression or tissue specificexpression); R (such as to remove TAR stem loops); or U5 (such as to useenhanced non-U5 based polyadenylation signals, for example from thebovine growth hormone gene).

[0031] In one configuration the internal promoter cassette is reversedand a polyadenylation signal is placed downstream of the cassette.

[0032] In another embodiment the polyadenylation signal which is usedcontains at least one intron.

[0033] The vector of the present invention may make use ofself-inactivating strategies. Self-inactivating retroviral vectors havebeen constructed by deleting the transcriptional enhancers or theenhancers and promoters in the U3 region of the 3′ LTR. After one roundof vector replication, these changes are copied into both the 5′ and the3′ LTRs producing an inactive provirus. However, any promoters internalto the LTRs in such vectors will still be active. This strategy has beenemployed to eliminate effects of the enhancers and promoters in theviral LTRs on transcription from internally placed genes. Such effectsinclude increased transcription or suppression of transcription. Thisstrategy can also be used to eliminate downstream transcription from the3′ LTR into genomic DNA. This is of particular concern in human genetherapy where it is of critical importance to prevent any activation ofan endogenous oncogene.

[0034] Another type of self-inactivating vector has been constructedthat has direct repeats flanking the packaging signal such that thepackaging signal is frequently deleted during reverse transcription,producing virus defective for packaging. With sufficiently long directrepeats, a majority of resultant proviruses lose their packagingsequences. The rate of deletion could be increased to 100% by designingthe vector so that packaging signal deletion reconstituted the neomarker nad be selecting the vector-infected cells in G418. This strategymay be particularly useful for gene therapy applications where anyspread of the vector following gene transfer is undesirable.

[0035] In a further preferred embodiment of the first aspect of theinvention, one or more nucleotides of interest (NOI) is introduced intothe vector at the cloning site. Such therapeutic genes may be expressedfrom a promoter placed in the retroviral LTR or may be expressed from aninternal promoter introduced at the cloning site.

[0036] Suitable NOI coding sequences include those that are oftherapeutic and/or diagnostic application such as, but are not limitedto: sequences encoding cytokines, chemokines, hormones, antibodies,engineered immunoglobulin-like molecules, a single chain antibody,fusion proteins, enzymes, immune co-stimulatory molecules,immunomodulatory molecules, anti-sense RNA, a transdominant negativemutant of a target protein, a toxin, a conditional toxin, an antigen, atumour suppressor protein and growth factors, membrane proteins,vasoactive proteins and peptides, anti-viral proteins and ribozymes, andderivatives therof (such as with an associated reporter group). Whenincluded, such coding sequences may be typically operatively linked to asuitable promoter, which may be a promoter driving expression of aribozyme(s), or a different promoter or promoters.

[0037] The NOI coding sequence may encode a fusion protein or a segmentof a coding sequence.

[0038] The retroviral vector of the present invention may be used todeliver a NOI such as a pro-drug activating enzyme to a tumour site forthe treatment of a cancer. In each case, a suitable pro-drug is used inthe treatment of the individual (such as a patient) in combination withthe appropriate pro-drug activating enzyme. An appropriate pro-drug isadministered in conjunction with the vector. Examples of pro-drugsinclude: etoposide phosphate (with alkaline phosphatase, Senter et al1988 Proc Natl Acad Sci 85: 4842-4846); 5-fluorocytosine (with cytosinedeaminase, Mullen et al 1994 Cancer Res 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerret al 1990 Cancer Immunol Immunother 31: 202-206);Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with β-lactamase);SR4233 (with P450 Reducase); Ganciclovir (with HSV thymidine kinase,Borrelli et al 1988 Proc Natl Acad Sci 85: 7572-7576); mustard pro-drugswith nitroreductase (Friedlos et al 1997 J Med Chem 40: 1270-1275) andCyclophosphamide (with P450 Chen et al 1996 Cancer Res 56: 1331-1340).

[0039] The vector of the present invention may be delivered to a targetsite by a viral or a non-viral vector.

[0040] As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Byway of example, some vectors used in recombinant DNA techniques allowentities, such as a segment of DNA (such as a heterologous DNA segment,such as a heterologous cDNA segment), to be transferred into a targetcell. Optionally, once within the target cell, the vector may then serveto maintain the heterologous DNA within the cell or may act as a unit ofDNA replication. Examples of vectors used in recombinant DNA techniquesinclude plasmids, chromosomes, artificial chromosomes or viruses.

[0041] Non-viral delivery systems include but are not limted to DNAtransfection methods. Here, transfection includes a process using anon-viral vector to deliver a gene to a target mammalian cell.

[0042] Typical transfection methods include electroporation, DNAbiolistics, lipid-mediated transfection, compacted DNA-mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationicagent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology1996 14; 556), and combinations thereof.

[0043] Viral delivery systems include but are not limited to adenovirusvector, an adeno-associated viral (AAV) vector, a herpes viral vector,retroviral vector, lentiviral vector, baculoviral vector. Other examplesof vectors include ex vivo delivery systems, which include but are notlimited to DNA transfection methods such as electroporation, DNAbiolistics, lipid-mediated transfection, compacted DNA-mediatedtransfection.

[0044] The term “retroviral vector particle” refers to the packagedretroviral vector, that is preferably capable of binding to and enteringtarget cells. The components of the particle, as already discussed forthe vector, may be modified with respect to the wild type retrovirus.For example, the Env proteins in the proteinaceous coat of the particlemay be genetically modified in order to alter their targetingspecificity or achieve some other desired function.

[0045] Preferably, the viral vector preferentially transduces a certaincell type or cell types.

[0046] More preferably, the viral vector is a targeted vector, that isit has a tissue tropism which is altered compared to the native virus,so that the vector is targeted to particular cells.

[0047] For retroviral vectors, this may be achieved by modifying the Envprotein. The Env protein of the retroviral secondary vector needs to bea non-toxic envelope or an envelope which may be produced in non-toxicamounts within the primary target cell, such as for example a MMLVamphotropic envelope or a modified amphotropic envelope. The safetyfeature in such a case is preferably the deletion of regions or sequencehomology between retroviral components.

[0048] Preferably the envelope is one which allows transduction of humancells. Examples of suitable env genes include, but are not limited to,VSV-G, a MLV amphotropic env such as the 4070A env, the-RD114 felineleukaemia virus env or haemagglutinin (HA) from an influenza virus. TheEnv protein may be one which is capable of binding to a receptor on alimited number of human cell types and may be an engineered envelopecontaining targeting moieties. The env and gag-pol coding sequences aretranscribed from a promoter and optionally an enhancer active in thechosen packaging cell line and the transcription unit is terminated by apolyadenylation signal. For example, if the packaging cell is a humancell, a suitable promoter-enhancer combination is that from the humancytomegalovirus major immediate early (hCMV-MIE) gene and apolyadenylation signal from SV40 virus may be used. Other suitablepromoters and polyadenylation signals are known in the art.

[0049] The packaging cell may be an in vivo packaging cell in the bodyof an individual to be treated-orit may be a cell cultured in vitro suchas a tissue culture cell line. Suitable cell lines include mammaliancells such as murine fibroblast derived cell lines or human cell lines.Preferably the packaging cell line is a human cell line, such as forexample: 293 cell line, HEK293, 293-T, TE671, HT1080.

[0050] Alternatively, the packaging cell may be a cell derived from theindividual to be treated such as a monocyte, macrophage, stem cells,blood cell or fibroblast. The cell may be isolated from an individualand the packaging and vector components administered ex vivo followed byre-administration of the autologous packaging cells. Alternatively thepackaging and vector components may be administered to the packagingcell in vivo. Methods for introducing retroviral packaging and vectorcomponents into cells of an individual are known in the art. Forexample, one approach is to introduce the different DNA sequences thatare required to produce a retroviral vector particle e.g. the env codingsequence, the gag-pol coding sequence and the defective retroviralgenome into the cell simultaneously by transient triple transfection(Landau & Littman 1992 J. Virol. 66, 5110; Soneoka et al 1995 NucleicAcids Res 23:628-633).

[0051] In one embodiment the vector configurations of the presentinvention use as their production system, three transcription unitsexpressing a genome, the gag-pol components and an envelope. Theenvelope expression cassette may include one of a number of envelopessuch as VSV-G or various murine retrovirus envelopes such as 4070A.

[0052] Conventionally these three cassettes would be expressed fromthree plasmids transiently transfected into an appropriate cell linesuch as 293T or from integrated copies in a stable producer cell line.An alternative approach is to use another virus as an expression systemfor the three cassettes, for example baculovirus or adenovirus. Theseare both nuclear expression systems. To date the use of a poxvirus toexpress all of the components of a retroviral or lentiviral vectorsystem has not been described. In particular, given the unusual codonusage of lentiviruses and their requirement for RNA handling systemssuch as the rev/RRE system it has not been clear whether incorporationof all three cassettes and their subsequent expression in a vector thatexpresses in the cytoplasm rather than the nucleus is feasible. Untilnow the possibility remained that key nuclear factors and nuclear RNAhandling pathways would be required for expression of the vectorcomponents and their function in the gene delivery vehicle. Here wedescribe such a system and show that lentiviral components can be madein the cytoplasm and that they assemble into functional gene deliverysystems. The advantage of this system is the ease with which poxvirusescan be handled, the high expression levels and the ability to retainintrons in the vector genomes.

[0053] According to another aspect therefore there is provided a hybridviral vector system for in vivo gene delivery, which system comprises aprimary viral vector which is obtainable from or is based on a poxvirusand a second viral vector which is obtainable from or is based on avectroviral vector, preferably a lentiviral vector, even more preferablya non-primate lentiviral vector.

[0054] The secondary vector may be produced from expression of essentialgenes for retroviral vector production encoded in the DNA of the primaryvector. Such genes may include a gag-pol from a retrovirus, an env genefrom an enveloped virus and a defective retroviral vector containing oneor more therapeutic or diagnostic NOI(s). The defective retroviralvector contains in general terms sequences to enable reversetranscription, at least part of a 5′ long terminal repeat (LTR), atleast part of a 3′LTR and a packaging signal.

[0055] If it is desired to render the secondary vector replicationdefective, that secondary vector may be encoded by a plurality oftranscription units, which may be located in a single or in two or moreadenoviral or other primary vectors.

[0056] In some therapeutic or experimental situations it may bedesirable to obviate the need to make EAIV derived from MVA in vitro.MVA-EIAV hybrids are delivered directly into the patient/animal e.g.MVA-EIAV is injected intravenously into the tail vein of a mouse andthis recombinant virus infects a variety of murine tissues e.g. lung,spleen etc. Infected cells express transduction competent EIAVcontaining a therapeutic gene for gene therapy for example. EIAV vectorparticles bud from these cells and transduce neighbouring cells. Thetransduced cell then contains an integrated copy of the EIAV vectorgenome and expresses the therapeutic gene product or other gene productof interest. If expression of the therapeutic gene product ispotentially toxic to the host it may be regulated by a specificpromoter, e.g. the hypoxic response element (HRE), which will restrictexpression to those cells in a hypoxic environment. For gene therapy oflung/trachea epithelium cells e.g to treat cystic fibrosis MVA-EIAV maybe given as an aerosol delivered intranasally. Alternatively,macrophages can be transduced in vitro and then reintroduced to createmacrophage factories for EIAV-based vectors. Furthermore, because MVA isreplication incompetent MVA-EIAV hybrids could also be used to treatimmuno-suppressed hosts.

[0057] Vaccinia virus, the prototypic member of the orthopox genuswithin the family poxviridae, was the first virus used for expression ofrecombinant exogenous proteins (Mackett et al 1982, Paoletti & Panicalli1982). Vaccinia virus has a large DNA genome of greater than 180 kb andreports indicate that it can accommodate over 25 kb of foreign DNA(Merchlinsky & Moss 1992). Several other strains of poxviruses have beenadapted as recombinant expression vectors (for review see Carroll andMoss 1997) e.g. fowlpox (Taylor & Paoletti 1988), canarypox (Taylor etal 1991), swinepox (van der Leek et al 1994) and entomopox (Li et al1997). Additionally, due to safety concerns, several highly attenuatedstrains of vaccinia virus have been developed that are compromised inhuman and other mammalian cells e.g. modified vaccinia virus Ankara(MVA) (Mayr 1978, Sutter 1992), NYVAC (Paoletti et al 1994), vacciniavirus deficient in a DNA replication enzyme (Holzer et al 1997). Thesemay all be used in the present invention.

[0058] MVA was derived from a replication competent vaccinia smallpoxvaccine strain, Ankara. After >500 passages in chick embryo fibroblastcells the virus isolate was shown to be highly attenuated in a number ofanimal models including mice that were immune deficient (Mayr et al1978). The attenuated isolate, MVA, was used to vaccinate over 120,000people, many of which were immunocompromised (Mahnel 1994) withoutadverse effects. Studies illustrate that MVA can infect a wide range ofmammalian cells but productive infection has only been observed inHamster kidney cell BHK-21 (Carroll 1997). In all other tested mammaliancell lines early expression, DNA replication and late expression areobserved leading to the production of non-infectious immature virusparticles (Carroll 1997, Meyer 1991). Virus replication studies showthat a minority of mammalian cells can support very low level productionof infectious virus i.e. BS-C-1 cells in which 1 infectious MVA particleis produced per cell (Carroll and Moss 1997). Late gene expressionusually give rise to >10 fold more protein that those genes under earlypromoters (Chakrabarti et al 1997, Wyatt et al 1996). In all otherattenuated poxvirus strains late gene expression is rarely observed inmammalian cells.

[0059] Production of retrovirus vector systems e.g. MLV-HIV andlentivirus vector systems requires the construction of producer linesthat express the virus genome and essential structural proteins to maketransduction competent virus. Generally, this is a relativelyinefficient process which is further complicated when the virus ispseudotyped with toxic envelope proteins such as VSV-G. Expression of afunctional genome and the required structural proteins from within arecombinant poxvirus may obviate many of the current inefficientretrovirus and lentivirus vector production technologies. Additionally,such recombinant poxviruses may be directly injected into patients togive rise to in vivo production of retrovirus or lentivirus.

[0060] MVA is a particularly suitable poxvirus for the construction of apox-retrovirus or pox-lentivirus hybrid due to its non-replicatingphenotype and its ability to perform both early and strong lateexpression for the production of high titre vector preparations.

[0061] In order to produce a functional retrovirus or lentivirus vectorgenome it is essential that the 5′ of the RNA genome should be exact(Cannon et al. 1996). This is a challenge in a vaccinia-based productionsystem as many of the vaccinia promoter comprise downstream determinantsof transcription efficiency (Davison 1989b, Moss 1996). However, we showthat thereare several ways to solve this problem:

[0062] a. Use of a T7 promoter and T7 termination sequence.

[0063] b. Use of early promoters (in which sequences downstream of theRNA start site are not highly conserved), (Davison 1989a).

[0064] c. Use of intermediate and late promoters of vaccinia whichrequire additional sequences downstream of the initiation site inconjunction with strategies to generate an authentic 5′ end or whichplace the additional downstream sequences into both R regions. There isa requirement for specific sequences of 4 nucleotides downstream of theinitiation of transcription in the late promoter (Davison 1989b, Moss1996). In the first case a ribozyme is placed downstream of the promoterand upstream of the R region. The ribozyme is designed to cleave the RNAin cis to generate the correct 5′ end. In the second the approach is tomodify the R regions to incorporate the extra sequences. This must bedone in both the 5′ and the 3′ LTR R regions.

[0065] The advantage of having a T7 dependent system is that it wouldrequire the infection of the cell by two recombinant vaccinia viruses toproduce transducing EIAV viral particles. For example, one MVA couldcarry the vector genome, under the control of the T7 promoter and thegag/pol and the env sequences under the control of the vacciniapromoters. The other MVA would carry the T7 polymerase gene under thecontrol of a vaccinia promoter (Wyatt et al 1995).

[0066] The retroviral vector particle according to the invention willalso be capable of transducing cells which are slowly-dividing, andwhich non-lentiviruses such as MLV would not be able to efficientlytransduce. Slowly-dividing cells divide once in about every three tofour days including certain tumour cells. Although tumours containrapidly dividing cells, some tumour cells especially those in the centreof the tumour, divide infrequently. Alternatively the target cell may bea growth-arrested cell capable of undergoing cell division such as acell in a central portion of a tumour mass or a stem cell such as ahaematopoietic stem cell or a CD34-positive cell. As a furtheralternative, the target cell may be a precursor of a differentiated cellsuch as a monocyte precursor, a CD33-positive cell, or a myeloidprecursor. As a further alternative, the target cell may be adifferentiated cell such as a neuron, astrocyte, glial cell, microglialcell, macrophage, monocyte, epithelial cell, endothelial cell orhepatocyte. Target cells may be transduced either in vitro afterisolation from a human individual or may be transduced directly in vivo.

[0067] The delivery of one or more therapeutic genes by a vector systemaccording to the present invention may be used alone or in combinationwith other treatments or components of the treatment.

[0068] For example, the retroviral vector of the present invention maybe used to deliver one or more NOI(s) useful in the treatment of thedisorders listed in WO-A-98/05635. For ease of reference, part of thatlist is now provided: cancer, inflammation or inflammatory disease,dermatological disorders, fever, cardiovascular effects, haemorrhage,coagulation and acute phase response, cachexia, anorexia, acuteinfection, HIV infection, shock states, graft-versus-host reactions,autoimmune disease, reperfusion injury, meningitis, migraine andaspirin-dependent anti-thrombosis; tumour growth, invasion and spread,angiogenesis, metastases, malignant, ascites and malignant pleuraleffusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis,rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis,neurodegeneration, Alzheimer's disease, atherosclerosis, stroke,vasculitis, Crohn's disease and ulcerative colitis; periodontitis,gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysisbullosa; corneal ulceration, retinopathy and surgical wound healing;rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis,congestive heart failure, endometriosis, atherosclerosis orendosclerosis.

[0069] In addition, or in the alternative, the retroviral vector of thepresent invention may be used to deliver one or more NOI(s) useful inthe treatment of disorders listed in WO-A-98/07859. For ease ofreference, part of that list is now provided: cytokine and cellproliferation/differentiation activity; immunosuppressant orimmunostimulant activity (e.g. for treating immune deficiency, includinginfection with human immune deficiency virus; regulation of lymphocytegrowth; treating cancer and many autoimmune diseases, and to preventtransplant rejection or induce tumour immunity); regulation ofhaematopoiesis, e.g. treatment of myeloid or lymphoid diseases;promoting growth of bone, cartilage, tendon, ligament and nerve tissue,e.g. for healing wounds, treatment of burns, ulcers and periodontaldisease and neurodegeneration; inhibition or activation offollicle-stimulating hormone (modulation of fertility);chemotactic/chemokinetic activity (e.g. for mobilising specific celltypes to sites of injury or infection); haemostatic and thrombolyticactivity (e.g. for treating haemophilia and stroke); antiinflammatoryactivity (for treating e.g. septic shock or Crohn's disease); asantimicrobials; modulators of e.g. metabolism or behaviour; asanalgesics; treating specific deficiency disorders; in treatment of e.g.psoriasis, in human or veterinary medicine.

[0070] In addition, or in the alternative, the retroviral vector of thepresent invention may be used to deliver one or more NOI(s) useful inthe treatment of disorders listed in WO-A-98/09985. For ease ofreference, part of that list is now provided: macrophage inhibitoryand/or T cell inhibitory activity and thus, anti-inflammatory activity;anti-immune activity, i.e. inhibitory effects against a cellular and/orhumoral immune response, including a response not associated withinflammation; inhibit the ability of macrophages and T cells to adhereto extracellular matrix components and fibronectin, as well asup-regulated fas receptor expression in T cells; inhibit unwanted immunereaction and inflammation including arthritis, including rheumatoidarthritis, inflammation associated with hypersensitivity, allergicreactions, asthma, systemic lupus erythematosus, collagen diseases andother autoimmune diseases, inflammation associated with atherosclerosis,arteriosclerosis, atherosclerotic heart disease, reperfusion injury,cardiac arrest, myocardial infarction, vascular inflammatory disorders,respiratory distress syndrome or other cardiopulmonary diseases,inflammation associated with peptic ulcer, ulcerative colitis and otherdiseases of the gastrointestinal tract, hepatic fibrosis, livercirrhosis or other hepatic diseases, thyroiditis or other glandulardiseases, glomerulonephritis or other renal and urologic diseases,otitis or other oto-rhino-laryngological diseases, dermatitis or otherdermal diseases, periodontal diseases or other dental diseases, orchitisor epididimo-orchitis, infertility, orchidal trauma or otherimmune-related testicular diseases, placental dysfunction, placentalinsufficiency, habitual abortion, eclampsia, pre-eclampsia and otherimmune and/or inflammatory-related gynaecological diseases, posterioruveitis, intermediate uveitis, anterior uveitis, conjunctivitis,chorioretinitis, uveoretinitis, optic neuritis, intraocularinflammation, e.g. retinitis or cystoid macular oedema, sympatheticophthalmia, scleritis, retinitis pigmentosa, immune and inflammatorycomponents of degenerative fondus disease, inflammatory components ofocular trauma, ocular inflammation caused by infection, proliferativevitreo-retinopathies, acute ischaemic optic neuropathy, excessivescarring, e.g. following glaucoma filtration operation, immune and/orinflammation reaction against ocular implants and other immune andinflammatory-related ophthalmic diseases, inflammation associated withautoimmune diseases or conditions or disorders where, both in thecentral nervous system (CNS) or in any other organ, immune and/orinflammation suppression would be beneficial, Parkinson's disease,complication and/or side effects from treatment of Parkinson's disease,AIDS-related dementia complex HIV-related encephalopathy, Devic'sdisease, Sydenham chorea, Alzheimer's disease and other degenerativediseases, conditions or disorders of the CNS, inflammatory components ofstokes, post-polio syndrome, immune and inflammatory components ofpsychiatric disorders, myelitis, encephalitis, subacute sclerosingpan-encephalitis, encephalomyelitis, acute neuropathy, subacuteneuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora,myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington'sdisease, amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.

[0071] The present invention also provides a pharmaceutical compositionfor treating an individual by gene therapy, wherein the compositioncomprises a therapeutically effective amount of the retroviral vector ofthe present invention comprising one or more deliverable therapeuticand/or diagnostic NOI(s) or a viral particle produced by or obtainedfrom same. The pharmaceutical composition may be for human or animalusage. Typically, a physician will determine the actual dosage whichwill be most suitable for an individual subject and it will vary withthe age, weight and response of the particular individual.

[0072] The composition may optionally comprise a pharmaceuticallyacceptable carrier, diluent, excipient or adjuvant. The choice ofpharmaceutical carrier, excipient or diluent can be selected with regardto the intended route of administration and standard pharmaceuticalpractice. The pharmaceutical compositions may comprise as—or in additionto—the carrier, excipient or diluent any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), solubilisingagent(s), and other carrier agents that may aid or increase the viralentry into the target site (such as for example a lipid deliverysystem).

[0073] Where appropriate, the pharmaceutical compositions can beadministered by any one or more of: inhalation, in the form of asuppository or pessary, topically in the form of a lotion, solution,cream, ointment or dusting powder, by use of a skin patch, orally in theform of tablets containing excipients such as starch or lactose, or incapsules or ovules either alone or in admixture with excipients, or inthe form of elixirs, solutions or suspensions containing flavouring orcolouring agents, or they can be injected parenterally, for exampleintracavemosally, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions may be best used in the formof a sterile aqueous solution which may contain other substances, forexample enough salts or monosaccharides to make the solution isotonicwith blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can beformulated in a conventional manner.

[0074] The delivery of one or more therapeutic genes by a vector systemaccording to the invention may be used alone or in combination withother treatments or components of the treatment. Diseases which may betreated include, but are not limited to: cancer, neurological diseases,inherited diseases, heart disease, stroke, arthritis, viral infectionsand diseases of the immune system. Suitable therapeutic genes includethose coding for tumour suppressor proteins, enzymes, pro-drugactivating enzymes, immunomodulatory molecules, antibodies, engineeredimmunoglobulin-like molecules, fusion proteins, hormones, membraneproteins, vasoactive proteins or peptides, cytokines, chemokines,anti-viral proteins, antisense RNA and ribozymes.

[0075] In a preferred embodiment of a method of treatment according tothe invention, a gene encoding a pro-drug activating enzyme is deliveredto a tumour using the vector system of the invention and the individualis subsequently treated with an appropriate pro-drug. Examples ofpro-drugs include etoposide phosphate (used with alkaline phosphataseSenter et al., 1988 Proc. Natl. Acad. Sci. 85: 4842-4846);5-fluorocytosine (with Cytosine deaminase Mullen et al. 1994 Cancer Res.54: 1503-1506); Doxorubicin-N-p-hydroxyphenoxyacetamide (withPenicillin-V-Amidase (Kerr et al. 1990 Cancer Immunol. Immunother. 31:202-206); Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (withCarboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (withb-lactamase); SR4233 (with P450 Reducase); Ganciclovir (with HSVthymidine kinase, Borrelli et al. 1988 Proc. Natl. Acad. Sci. 85:7572-7576) mustard pro-drugs with nitroreductase (Friedlos et al. 1997JMed Chem 40: 1270-1275) and Cyclophosphamide or Ifosfamide (with acytochrome P450 Chen et al. 1996 Cancer Res 56: 1331-1340).

[0076] In accordance with the invention, standard molecular biologytechniques may be used which are within the level of skill in the art.Such techniques are fully described in the literature. See for example;Sambrook et al (1989) Molecular Cloning; a laboratory manual; Hames andGlover (1985-1997) DNA Cloning: a practical approach, Volumes I-IV(second edition); Methods for the engineering of immunoglobulin genesare given in McCafferty et al (1996) “Antibody Engineering: A PracticalApproach”.

[0077] The invention will now be further described by way of example inwhich reference is made to the following Figures in which:

[0078]FIG. 1—shows the structure of transcription units from plasmidspESP, pONY3 and pONY2.lnlsLacZ.

[0079]FIG. 2—shows a PCR analysis of integrated EIAV vector. PCR wasperformed with either genomic DNA from EIAV vector transduced cells(lanes 1 and 5) or mock transduced cells (lanes 2 and 6). pONY2.lnlsLacZ(lanes 3 and 7) and pONY3 (lanes 4 and 8) were used as controls. A. PCRdetection of EIAV LTRs. B. PCR detection of pol.

[0080]FIG. 3—shows the structure of vector transcription units indeletion plasmids used to identify the packaging requirements for anEIAV vector.

[0081]FIG. 4—shows a secondary structure prediction for the RNA derivedfrom the gag-transcription unit present in pONY2.13LacZ.

[0082]FIG. 5 is a representation of vectors derived from the EIAVgenome.

[0083]FIG. 6 is a representation of gagpol constructs derived from EIAV.

[0084]FIG. 7 is a representation of an EIAV vector comprising an S2deletion

[0085]FIG. 8 is a representation of EIAV gagpol constructs havingdeleted S2 and dUTPase genes.

[0086]FIG. 9 is a representation of an EIAV minimal vector.

[0087]FIG. 10 illustrates a gel showing an analysis of EIAV gagpolconstructs according to the invention.

[0088]FIG. 11 shows examples of the pONY4 vectors.

[0089]FIG. 12 shows two SIN vectors.

[0090]FIG. 13 is a representation of a vector with a split polyA signal.

[0091]FIG. 14 is a representation of a vector with a split polyA signal.

[0092]FIG. 15 is a representation of a vector with a split polyA signal.

[0093]FIG. 16 shows construction of pONY4-GFP with a split polyA signal.

[0094]FIG. 17 shows construction of a MLV/EIAV vector.

[0095]FIG. 18 shows primers for construction of MLV/EIAV vectors.

[0096]FIG. 19 shows complete sequence of pONY-mouse.

[0097]FIGS. 20 and 21 give PCR priming.

[0098]FIG. 22 shows pEMVA4 (after PCR with primers EMVA 1-8).

[0099]FIG. 23 shows pEMVA4.

[0100]FIG. 24 shows pEMVA5.

[0101]FIGS. 25 and 26 show an example of hammer-head strategy for 5′ endformation.

[0102]FIG. 27 shows pEMVA6.

[0103]FIG. 28 shows pEMVA7 and pSyn pONY4.1.

[0104]FIG. 29 shows EMVA 10/11.

[0105]FIG. 30 shows pEMVA9.

[0106]FIG. 31 shows pEMVA10.

[0107]FIG. 32 shows pLWHORSE3.1.

[0108]FIG. 33 shows pMCRev.

[0109]FIG. 34 shows pYFVSVG.

[0110]FIG. 35 shows pYFAmpho.

[0111]FIG. 36 shows recombinant MVA constructs.

[0112]FIG. 37 shows the complete sequence of pSC65.

[0113]FIG. 38 shows the complete sequence of pLW22

[0114] In more detail, FIG. 1. Plasmids used in this study. The genomicorganization of EIAV is indicated including splice donor (d1, d2 and d3)and splice acceptor sites (a1, a2 and a3). The positions of gag, pol,env, tat, rev, S2 and the viral LTRs are also shown. Plasmid pESP is anEIAV vector genome containing the SV40 promoter and the puromycinresistance gene. Plasmid pONY3 is an EIAV gagpol expression plasmid.pONY2.lnlslacZ is an EIAV vector genome containing a HCMV IEenhancer/promoter and β-galactosidase gene (nlslacZ).

[0115]FIG. 2 shows PCR analysis of integrated EIAV vector. PCR wasperformed with either genomic DNA from EIAV vector transduced cells(lanes 1 and 5) or mock transduced cells (lanes 2 and 6). pONY2.lnlsLacZ(lanes 3 and 7) and pONY3 (lanes 4 and 8) were used as controls. A. PCRdetection of EIAV LTRs. B. PCR detection of pol.

[0116] TABLE 2. Transduction of dividing and non-dividing cells. 293Tcells were treated with (open columns) or without (shaded columns)aphidicolin according to the method of Lewis et al. (Lewis, P. F., andM. Emerman. 1994. J Virol. 68:510-6.) and then transduced with eitherEIAV vector PONY2.lnlsLacZ or MLV vector HIT111 (Soneoka et al 1995Nucl. Acids Res. 23:628-633). 48 hours later the cells were stained withX-gal. Titers were averaged from two independent experiments andcalculated as lac Z colony forming units per ml. There was no more than10% variation between experiments.

[0117]FIG. 10 shows analysis of gagpol expression constructs. 30%lg oftotal cellular protein was separated by SDS/Page electrophoresis,transferred to nitro-cellulose and probed with anti-EIAV antibodies. Thesecondary antibody was anti-Horse HRP (Sigma). Titres were averaged fromthree independent experiments and calulated as lacZ forming units perml. There was no more than 10% variation between experiments.pONY2.lnlslacZ and the envelope expression plasmids were co-transfectedwith the EIAV gagpol.

EXAMPLE 1 Construction of EIAV Vectors Containing Deleted Gag Genes

[0118] In order to construct a replication incompetent EIAV vectorsystem we have used, as a starting point, an infectious proviral clonepSPEIAV19 (accession number: U01866), described by Payne et al. (1994, JGen Virol. 75:425-9). An initial EIAV based vector was constructed bysimply deleting part of env by removing a Hind III/Hind III fragmentcorresponding to coordinates 5835/6571 according to the numbering systemof Payne et al. (ibid.). This fragment was replaced with the puromycinresistance gene under the control of the SV40 early promoter frompTIN500 (Cannon et al 1996 J. Virol. 70:8234-8240) to create pESP (FIG.1). Viral stocks were produced by calcium phosphate transfection of 293Tcells (Soneoka et al 1995 Nucl. Acids Res. 23:628-633) with pESP andpRV67 (Kim et al 1998 J. Virol. 72(1):811-6) a plasmid in which thevesicular stomatitis virus glycoprotein (VSV-G) is expressed from theHCMV-IE enhancer/promoter. Alternatively other VSV-G expression plasmidscan be used eg Yee, et al 1994 PNAS 91:9564-9568. The resultingsupernatant was used to transduce human kidney (293T) and canineosteosarcoma cells (D17) as follows. 48 hours post-transfection tissueculture fluid was collected and filtered through 0.45 cm filters.Ten-fold dilutions were made in culture medium containing polybrene at 8μg/ml and then 500 μl aliquots placed on D17 cells seeded at1.6×10⁵/well in 12 well plates on the previous day. Two hours later 1 mlof culture media was added. Two days later puromycin was added to afinal concentration of 4 ug/ml and incubation was continued for afurther 7 days. As a positive control, a Murine leukaemia virus (MLV)based vector (pTIN500) containing the puromycin resistance gene underthe control of the SV40 early promoter was used in conjunction withpHIT60 (MLV gagpol) and pRV67 (Cannon et al 1996 J. Virol.70:8234-8240). No resistance colonies were detected on either cell typeafter 7 days of puromycin selection with the EIAV vector. The MLV vectorproduced 5.0×10⁴ c.f.u./ml on 293T cells and 1.0×10⁴ c.f.u./ml on D17cells.

[0119] The likely explanation for this result is that the EIAV LTR isnot functional in human cells in the absence of tat and so insufficientamounts of the critical components such as gag-pol and tat are produced.

[0120] A further vector system was therefore constructed comprisingthree transcription units to produce the following: 1) vector genomeRNA; 2) env and 3) gag-pol. In order to ensure that sufficient of eachcomponent is produced, the env and gag-pol transcription units aretranscribed from a promoter-enhancer active in the chosen humanpackaging cell line. In this way, sufficient gag-pol and, most likelytat, are produced to ensure efficient production oftransduction-competent vector particles.

[0121] The vector genome was constructed which has the reporter genewithin the pol region of the genome as follows. The plasmid designatedpONYl was constructed by inserting the EIAV LTR, amplified by PCR frompSPEIAV19, into pBluescript II KS+(Stratagene). The 5′ LTR of EIAV clonepSPEIAV19 was PCR amplified using pfu polymerase with the followingprimers:

[0122] 5′ GCATGGACCTGTGGGGTTTTTATGAGG

[0123] 3′ GCATGAGCTCTGTAGGATCTCGAACAGAC

[0124] The amplicon was blunt ended by 5′ overhang fill-in and insertedinto pBluescript II KS+cut with Bss HII which had been blunt ended by 3′overhand removal using T4 DNA polymerase. This construct was calledpONY1 and the orientation was 5′ to 3′ in relation to β-galactosidase ofpBluescript II KS+. Sequencing of pONY1 revealed no mutations.

[0125] Vector genome pSPEIAV19DH was cut with Mlu I (216/8124) andinserted into pONY1 Mlu I cut (216) to make pONY2. A Bss HII digest(619/792) of pBluescript II KS+ was carried out to obtain the multiplecloning site. This was blunt ended by 5′ overhang fill-in and ligated topONY2 cut with Bgl II and Nco I (1901/4949) and blunt ended by 5′overhang fill-in. The orientation was 3′ to 5′ in relation to the EIAVsequence. This was called pONY2.1. pSPCMV was created by inserting pLNCX(Accession number: M28246) (Pst I/Hind III) into pSP72 (Promega). Theβ-galactosidase gene was inserted from pTIN414 (Cannon PM et al J.Virol. 70, 8234-8240) into pSP72 (Xho I/Sph I) to make pSPlacZ. The 5′end to the β-galactosidase gene was replaced by the SV40 T antigennuclear localization signal from pAD.RSVbgal (J. Clin. Invet.90:626-630, 1992). pAD.RSVbgal was cut with Xho I/Cla I and insertedinto Xho I/Cla I pSPlacZ to make pSPnlslacZ. The CMV nuclear localizingand non nuclear localizing β-galactosidase from pSPlacZ and pSPnlslacZwas cut out with Pst I and inserted into the Pst I site of pONY2.1 inthe 5′ to 3′ orientation of EIAV. These were called pONY2. lnlslacZ andpONY2.1lacZ.

[0126] An EIAV gagpol expression plasmid (pONY3) was then made byinserting Mlu I/Mlu I fragment from pONY²ΔH into the mammalianexpression plasmid pCl-neo (Promega) such that the gag-pol gene isexpressed from the hCMV-MIE promoter-enhancer. In particular, gagpolpSPEIAV19DH was cut with Mlu I (216/8124) and inserted into pCI-Neo(Promega) Mlu I cut (216) to make pONY3. Plasmid pONY3 should notproduce a functional genome because it lacks the appropriate LTRsequences. Virus was produced by transient three plasmid cotransfectionof 293T cells with pRV67, pONY3 and pONY2.10nlsLacZ as described forMLV-based vectors (Soneoka et al 1995 Nucl. Acids Res. 23:628-633) andthen used to transduce 293T cells and D17 cells as follows. 48 hourspost-transfection tissue culture fluid was collected and filteredthrough 0.45,um filters. Ten-fold dilutions were made in culture mediumcontaining polybrene at 8 μg/ml and then 500 ul aliquots placed on D17cells seeded at 1.6×10⁵/well in 12 well plates on the previous day. Twohours later 1 ml of culture media was added and incubation continued for48 hours prior to assessment of LacZ gene expression using the X-galstaining procedure. for E coli β-galactosidase (macGregor et al 1991Methods in Molecular Biology Vol 7 ed EJ Murray p217-235). In both casesthe virus transduced the cells at frequencies of about 10⁵LacZ-transducing cell—forming—unit (i.f.u.)/ml which was about 10-foldless than with the MLV-based vector produced from pH111. These datashowed that we had produced an EIAV-based vector system and alsosuggested that replacement of the Hind III/Hind III fragment in env withforeign DNA may disrupt the function of the genome.

[0127] We next characterized the ability of the EIAV vector particles tobe pseudotyped with envelope proteins from other viruses.pONY2.10nlsLacZ and pONY3 were cotranfected with envelope expressionplasmids producing MLV amphotropic (pHIT456) and MLV ecotropic (pHIT123)envelopes (Soneoka et al 1995 Nucl. Acids Res. 23:628-633) as well asVSV-G (pRV67) (Table 1). pHIT111 (MLV vector genome) and pHIT60 (MLVgagpol expression plasmid) were cotransfected with the envelope plasmidsas positive controls (Table 1). The viral supernatants were used totransduce a variety of cell lines including human kidney (293T), murineembryo (NIH3T3) and canine osteosarcoma (D17). As expected the celltropism of the virus was largely determined by the envelope. EIAV couldbe pseudotyped with amphotropic envelope, but transduction efficienciesvaried. The amphotropic pseudotyped virus gave titres of about 102 onD17 cells, 103 on NIH3T3 cells and 104 on 293T cells. The reason forthese differences was not pursued. EIAV could also be pseudotyped withthe MLV ecotropic envelope and these viruses transduced NIH3T3 cells attitres of 104 l.f.u./ml. EIAV, pseudotyped with VSV-G envelope,transduced all the cell lines tested. The titer varied between thedifferent envelopes and cell types but overall efficiencies wererelatively high for the non-murine cells, but still lower than with amurine vector system. Taken together, these data show that the EIAVvector system is not dependent on the EIAV envelope and can beeffectively pseudotyped with three envelopes conferring broad hostrange. This makes this system as generally useful as current MLV-basedsystems.

[0128] EIAV vectors can also be pseudotyped in the same manner using theRD114 envelope, for instance using pRDF (Cosset et al 1995 J. Virol. 69:7430-7436).

[0129] In order to characterize the transduction events further wecarried out a PCR analysis of 293T cells transduced by the EIAV vectorpseudotyped with VSV-G. In particular we asked if the vector genome, asopposed to a recombinant with the gagpol expression plasmid, pONY3, hadbeen the transduction vehicle for the β-galactosidase gene. PCRamplification using primers specific for the EIAV LTR gave the expectedPCR product of 310 bp when genomic DNA isolated from transduced cellswas used (FIG. 2A, lane 1). No PCR product was detected when mocktransduced 293T cell DNA was used as template (FIG. 2A, lane 2).pONY2.10nlsLacZ was used as a positive control (FIG. 2A, lane 3). No PCRproduct was detected when pONY3 was used as a template (FIG. 2A, lane4). The lack of a PCR product, when using pol specific primers, (FIG.2B) confirmed that no gagpol sequences from pONY3 had integrated intothe host chromosomes. Taken together these data show that the authenticvector genome had transduced the cells.

[0130] In order to determine if the EIAV vector retained the ability totransduce non-dividing cells, 293T cells were arrested in G1/S phase bytreatment with aphidicolin according to published procedures (Lewis andEmerman 1994) and then transduced with EIAV-based and MLV-based vectorspseudotyped with VSV-G. The transduction efficiency of the MLV vectorwas lower by four orders of magnitude in aphidicolin treated cells ascompared to untreated cells. The incomplete block to cell transductionby MLV was probably due to a small population of dividing cells. Incontrast, no significant difference was observed in the case of theEIAV-based vector. This demonstrates that the EIAV vector, like HIVvectors, can efficiently transduce non-dividing cells. The vector genomepONY2.10lacZ contains 1377 nt of gag. RNA secondary structure prediction(“http://www.ibc.wustl.edu/˜zuker/rna/”) was used to identify possiblestem-loop structures within the leader and the 5′ end of gag. Based onthese predictions four deletions were made within the gag region ofpONY2.10lacZ (FIG. 1). Deletions were made by PCR mutagenesis usingstandard techniques.

[0131] pONY2.1lacZ contains 1377 nt of gag (deleted from position 1901nt)

[0132] pONY2.11lacZ contains 354 nt of gag (deleted from position 878nt)

[0133] pONY2.12lacZ contains 184 nt of gag (deleted from position 708nt)

[0134] pONY2.13lacZ contains 109 nt of gag (deleted from position 633nt)

[0135] pONY2.14lacZ contains 2 nt of gag (deleted from position 526 nt)

[0136] These vectors were used in a three plasmid cotransfection asdescribed for MLV-based vectors (Soneoka et al 1995 Nucl. Acids Res.23:628-633) and the virus generated was titred on 293T and D17 cells.

[0137] It was found that the first 109 nt of gag coding sequence wereneeded for maximal packaging in addition to the un-translated region;pONY2.13lacz (Table 2). Similar titres were found on D17 cells. Thepredicted secondary structure of the gag sequence derived RNA inpONY2.13lacZ is shown in FIG. 4.

[0138] Based on the secondary structure prediction in FIG. 4, fourfurther deletions were made within the area upstream and downstream ofthe major splice donor codon in pONY2.13lacZ.

[0139] pONY2.2lacZ contains deleted between position 409 to 421 nt

[0140] pONY2.22lacZ contains deleted between position 424 to 463 nt

[0141] pONY2.23lacZ contains deleted between position 470 to 524 nt

[0142] pONY2.24lacZ contains deleted between position 529 to 582 nt

[0143] pONY2.25lacZ contains deleted between position 584 to 645 nt

[0144] pONY2.26lacZ contains deleted between position 409 to 421 nt andbetween position 470 to 542 nt.

[0145] These vectors were used in a three plasmid co-transfection asdescribed above and the virus generated was titred on D17 cells. It wasfound that deletions within this region severely affected the titre ofthe virus (Table 3). Constructs pONY2.23 and 2.26 gave the lowest titre.These both contained the deletion between position 470 to 524 nt. Theleast severe deletion was the one between position 409 to 421 nt. Basedon this information the region around the major splice donor is usefulfor optimal packaging.

[0146] Similar secondary structure predictions and deletion analysis maybe used to identify the packaging signal in other non-primatelentiviruses. TABLE 1 Transduction efficiency of viral vectors. Titer(l.f.u./ml)^(a) Vector Envelope D17 NIH3T3 293T pONY2.1nlslacZ Mock <1<1 <1 pONY2.1nlslacZ pHIT456 1.0 × 10² 8.4 × 10² 2.0 × 10⁴ (MLVamp)pONY2.1nlslacZ pHIT123 <1 1.5 × 10⁴ <1 (MLVeco) pONY2.1nlslacZ pRV67(VSVG) 1.0 × 10⁵ 3.6 × 10³ 2.0 × 10⁵ pHIT111 Mock <1 <1 <1 pHIT111pHIT456 1.3 × 10⁵ 2.6 × 10⁶ 2.0 × 10⁷ (MLVamp) pHIT111 pHIT123 <1 2.8 ×10⁶ <1 (MLVeco) pHIT111 pRV67 (VSVG) 3.0 × 10⁶ 2.0 × 10⁵ 5.0 × 10⁶

[0147] TABLE 2 Vector Titre Genome (l.f.u/ml) PONY2.10 3.30E + 04PONY2.11 1.60E + 05 PONY2.12 1.40E + 05 PONY2.13 1.70E + 05 PONY2.145.40E + 02 Mock  1.0E + 01

[0148] TABLE 3 Vector Titre Genome (l.f.u/ml) 2.21 1.20E + 04 2.223.80E + 03 2.23 1.20E + 02 2.24 5.20E + 02 2.25 5.60E + 02 2.26 1.00E +02 2.13 4.00E + 04

EXAMPLE 2 Construction of pEGASUS-1

[0149] An EIAV—based vector was made (pEGASUS-1) that contains only 759nt of EIAV sequences (268 nt-675 nt and 7942 nt-8292 nt) as follows.

[0150] Sequences encompassing the EIAV polypurine tract (PPT) and the3′LTR were obtained by PCR amplification from pONY2.10LacZ using primersPPTEIAV+(Y8198): GACTACGACTAGTGTATGTTTAGAAAAACAAGG, and 3′NEGSpeI(Y8199):CTAGGCTACTAGTACTGTAGGATCTCGAACAG. The product was purified,digested with SpeI (ACTAGT) and ligated into pBS II KS+ which had beenprepared by digestion with SpeI and treatment with alkaline phosphatase.Colonies obtained following transformation into E. coli, XL-1Blue werescreened for the presence of the 3′LTR in the orientation in which theU5 region of the 3′LTR was proximal to the NotI site of the pBS IIKS+linker. The sequence of the cloned insert was determined and showedthat it contained only one change from the EIAV clone pSPEIAV19 (AC:U01866). This was a ‘C’ insertion between bases 3 and 4 of the R region.The same change was found in the template used in the PCR reaction. Theclone was termed pBS.3′LTR.

[0151] Next the reporter gene cassette, CMV promoter/LacZ, wasintroduced into the PstI site of pBS.3′-LIR. The CMV/LacZ cassette wasobtained as a PstI fragment from pONY2.10LacZ (see above). The ligationreaction to join the above fragments was transformed into E. coli,XL-1Blue. A number of clones in which the CMV/LacZ insert was orientatedso that the LacZ gene was proximal to the 3′LTR were assessed foractivity of the CMV/LacZ cassette by transfection into the cell line293T using standard procedures. A clone which gave blue cells at 48hours post-transfection following development with X-gal was selectedfor further use and termed pBS CMVLacZ.3′LTR.

[0152] The 5′region of the EIAV vector was constructed in the expressionvector pCIEneo which is a derivative of pCIneo (Promega) modified by theinclusion of approximately 400 base pairs derived from the 5′end of thefull CMV promoter as defined previously. This 400 base pair fragment wasobtained by PCR amplifcation using primers VSAT1(GGGCTATATGAGATCTTGAATAATAAAATGTGT) and VSAT2 (TATTAATAACTAGT) andpHFT60 as template. The product was digested with BglII and SpeI andligated into pCIneo which had been digested similarly.

[0153] A fragment of the EIAV genome running from the R region to nt 150of the gag coding region (nt 268—to 675) was amplified with primersCMV5′EIAV2 (Z0591)(GCTACGCAGAGCTCGTTTAGTGAACCGGGCACTCAGATTCTG: and3′PSI.NEG (GCTGAGCTCTAGAGTCCTTTTCTTTTACAAAGTTGG) using as template DNA.The 5′region of the primer CMV5′EIAV2 contains the sequences immediatelyupstream of the CMV promoter transcriptional start site and can be cutwith SacI. 3′PSI.NEG binds 3′ of the EIAV packaging sequences as definedby deletion analysis (above) and contains an XbaI site. The PCR productwas trimmed with SacI and XbaI and ligated into pCIEneo which had beenprepared for ligation by digestion with the same enzymes. Thismanipulation places the start of the EIAV R region at thetranscriptional start point of the CMV promoter and transcripts producedthus start at the genuine start position used by EIAV and extend to the3′-side of the packaging signal. Clones which appeared to be correct asassessed by restriction analysis were sequenced. A clone termedpCIEneo.5′EIAV was selected for further work.

[0154] In the next step the CMVLacZ and 3′LTR cassette inpBS.CMVLacZ.3′LTR was introduced into pCIEneo.5′EIAV. pBS.CMVLacZ.3′LTRwas digested with ApaI, the 3′ overhangs removed with T4 DNA polymerase,then digested with NotI. The fragment containing the CMVLacZ.3′LTR waspurified by standard molecular biology techniques. The vector forligation with this fragment was prepared from pCIEneo.5′EIAV bydigestion with SalI, followed by filling-in of the 5′ overhangs using T4DNA polymerase. The DNA was then digested with NotI and purified priorto use in ligation reactions. Following transformation into E. coli,XL-1Blue colonies were screened for the presence of the insert byrestriction analysis to identify the required clone, designatedpEGASUS-1.

[0155] The function of the pEGASUS-1 EIAV vector was compared topONY2.10LacZ using the three plasmid co-transfection system as describedin Example 1. Comparable titres were obtained from both vectorsindicating that pEGASUS-1 contains all the sequences required forpackaging with good efficiency.

EXAMPLE 3 Introduction of RRE's into EIAV Vectors

[0156] Further improvements to the EIAV vector pEGASUS-1 may be made byintroduction of additional elements to improve titre. A convenient sitefor the introduction of such elements is the SalI site which liesbetween the XbaI to the 3′ of the packaging signal and upstream of theCMV/LacZ cassette of pEGASUS-1. For example the RRE from HV or EIAV canbe inserted at this site.

[0157] The HIV-1 RRE was obtained from the HIV-1 molecular clone pWI3(Kimpton and Emerman 1992 (J. Virol. 66: 2232-2239) by PCR amplificationusing primers RRE(+) GTCGCTGAGGTCGACAAGGCAAAGAGAAGAG and RRE(−)GACCGGTACCGTCGACAAGGCACAGCAGTGG. The fragment of DNA and pEGASUS-1 weredigested with SalI and following ligation, transformed into E. coli,XL-1 Blue. Colonies were screened for the presence of the HIV RRE andtwo clones, with the HIV RRE in either the positive or negativeorientation, used for further work These vectors, pEGASUS-2.HIV RRE(+)or pEGASUS-2.HIV RRE(−) can be tested in 293T cells by carrying out afour plasmid co-transfection in which the plasmid pCIneoHIVrev,expressing the rev protein from HIV-1 is co-transfected with vector,pONY3 and pRV67 plasmids

[0158] The EIAV RRE as defined previously (Martarano et al 1994) wasobtained by PCR amplification as follows. Using pONY2.10LacZ as template2 amplifications were performed to obtain the two parts of the EIAV RRE.The 5′-element was obtained using primers ERRE1(TTCTGTCGACGAATCCCAGGGGGAATCTCAAC) and ERRE2(GTCACCTTCCAGAGGGCCCTGGCTAAGCATAACAG) and the 3′element with ERRE3(CTGTTATGCTTAGCCAGGGCCCTCTGGAAGGTGAC) and ERRE4(AATTGCTGACCCCCAAAATAGCCATAAG). These products will anneal to each otherhence can be used in second PCR reaction to obtain a DNA which ‘encodes’the EIAV RRE. The PCR amplification is set up with out primers ERRE1 andERRE4 for the first 10 cycles and then these primers are added to thereaction and a further 10 cycles of amplification carried out. Theresulting PCR product and pEGASUS-1 were digested with SalI, ligated andtransformed into E. coli XL-1Blue. Clones in which the EIAV RRE was ineither the positive or negative orientations were selected for furtherwork. The activity of these vectors was assessed in 4-wayco-transfectioand pEGASUS-1 were digested with SalI, ligated andtransformed into E. coli XL-1Blue. Clones in which the EIAV RRE was ineither the positive or negative orientations were selected for furtherwork. The activity of these vectors was assessed in three palsmidco-transfections, (EIAV rev being supplied by pONY3) or in 4-plasmidco-transfection experiments as described above, but using pCIneo.EIAVRev to supply additional EIAV rev.

[0159] For construction of pCIneo EIAV REV the EIAV REV encodingsequences were derived by PCR amplification. The EIAV REV sequences wereobtained using a two step ‘overlapping’ PCR amplification procedure asdescribed above for the EIAV RRE. Template for the two reactions waspONY3 and primers for the 5′fragment were EIAV REV REV5′O(CCATGCACGTCTGCAGCCAGCATGGCAGAATCGAAG) and EAIV REV IN(CCTGAGGATCTATTTTCCACCAGTCATTTC) and for the 3′product EIAV REV IP(GTGGAAAATAGATCCTCAGGGCCCTCTGG) and EIAV.REV3′O(GCAGTGCCGGATCCTCATAAATGTTTCCTCCTTCG). The second PCR amplification wascarried out with primers EIAV REV5′O and EIAV REV3′O being added after10 cycles. The resulting product was ligated with the PCR fragment ‘TA’cloning vector pCR2.1 (Invitrogen) the orientation of the EIAV REVinsert was assessed by restriction enzyme analysis and the presence ofthe correct EIAV REV sequence confirmed. The construct was calledpTopoRevpos. The EIAV REV insert was excised from pTopoRevpos bydigestion with SpeI and NotI and ligated into pCIneo which had beendigested with NheI and NotI.

EXAMPLE 4 Transduction of Human Macrophages

[0160] Primary human monocytes were obtained from leukocyte-enrichedblood (from the National Blood Transfusion Service, Southmead RdBristol, UK) as follows. Peripheral blood mononuclear cells (PBMC) wereenriched by centrifugation above a Ficoll discontinuous gradient(Pharmacia) according to the manufacturer's instructions Macrophageswere obtained from this cell population by adherence to tissue cultureplastic over 7 days in RPM! 1640 medium (Dutch modified; Sigma)containing 2% heat-inactivated human AB serum (Sigma) or 10% FCS(Sigma). Non-adherent cells were removed by extensive washing of theplates with medium.

[0161] Virus for transduction experiments was obtained by three plasmidco-transfection into 293T cells. The vector for the experiments was apONY2.13 derivative in which the CMV/LacZ reporter cassette had beenreplaced with CMV/green fluorescent protein (GFP).

[0162] Vector pONY2.13GFP was made as follows. The sequence encoding thered-shifted GFP and eukaryotic translation signals was cut out ofpEGFP-N1 (Clontech “http://www.clontech.com/”) with BglII and XbaI andligated into the general cloning vector pSP72 (Promega) which had beenprepared by digestion with the same enzymes. The GFP-encoding sequenceswere then excised using XhoI and ligated into pONY2.13 which had beencut with XhoI (thereby releasing the LacZ coding region). Followingtransformation into E. coli, XL-1Blue clones in which the orientation ofthe GFP insert with respect to the CMV promoter was such that expressionwould be expected were determined restriction analysis and expression ofGFP confirmed by transfection of DNA into 293T cells.

[0163] Vector was recovered by three plasmid co-transfection into 293Tcells and harvested at 42-48 hours post-transfection: tissue culturefluid was 0.45(m-filtered and virus was then pelleted by centrifugationat 50,000 g (20 Krpm), for 90 minutes at 4(C in a SW40Ti rotor. Viruswas resupended in 50-100(1 of complete media for 2 hours and then usedin transduction experiments. Transductions with pONY2.13GFP vector werecarried out as follows. Macrophages, seeded at 5×10⁵ per well of 48-wellplates were washed once with medium and then 300 (l of medium was putback on the cells. Virus was added to the medium and gently pipetted up2-3 times to ensure mixing. Transduction efficiency was assessed at 3-5days post-transduction. The number of transduced macrophages wasdetermined using a fluorescence microscope. Expression of GFP can bemonitored for extended periods, e.g., up to several weeks.Alternatively, transductions can be carried out with vectors carryingthe LacZ marker. In such experiments the transduction frequency isassessed by detecting the presence of β-galactosidase usingimmunological procedures.

EXAMPLE 5 Introduction of EIAV Vectors in vivo in Rat Brain

[0164] Adult Wistar rats were anaesthetised with a solution containing 1part Nembutal (0.1 ml/35 gm body weight) 1 part Novetal (0.1 ml/35 gmbody weight) and 2 parts dH2O, and placed into a stereotaxic apparatus.A midline incision was made along the rostral-to-caudal length of thescalp and the skin deflected back to expose the skull. Using stereotaxiccoordinates (measured from Bregma) of 3.00 mm posterior and 3.00lateral, a 1 mm diameter hole was drilled into the skull. Unilateralintracortical injections of EIAV vectors were then made using a 10 μlHamilton syringe or a 1.0 μl fine glass capillary to various depths fromthe surface of the brain. The syringe was left in place an additional 5min to prevent reflux. Control animals receive a single 10 μlintracortical injection of saline with the Hamilton or 1.0 μl with thefine glass capillary. Animals were then sutured and left to recover.Forty-eight hours later, these animals were deeply anesthetized asdescribed above and perfused through the heart with 200 ml ofphosphate-buffered saline (PBS). The brains were then dissected out,frozen into dry-ice cooled isopentane (−30° C.) and cut coronally at 10μm with a cryostat. Every 5th section through the injection site and 2mm rostral and caudal are collected onto Super-Frost slides, fixed andeither X-gal or immunostained or stained with Cresyl Violet.

EXAMPLE 6 Transduction of Bronchial Cells Differentiated in Culture

[0165] Epithelial cells can be differentiated to form epithelia-likemonolayers which display (>1000 Ω cm³) electrical resistance and acuboidal morphology. There are various wasys to do this for exampleFuller et al 1984.This creates polarized cells. This polarity isfunctional and mimics epithelial cells in vivo. Thus EIAV vectors can beused to transduce these cells either through the basolateral surface orthe apical surface using vectors and preparations as described inExamples 1-3.

EXAMPLE 7 A Minimal EIAV System

[0166] In order to eliminate the risk of accessory genes or codingsequences having deleterious effects in therapeutic applications, vectorsystems lacking tat, S2 and the dUTPase are constructed.

[0167] Construction of S2 Mutants

[0168] A) Vector Genome

[0169] pONY2.13lacZ contains 109 nt of gag (deleted from nucleotidepositions 633 to 4949) (pONY2.13lacZ is described above). This vector isused to make an EIAV vector genome from which S2 expression iseliminated by deletion from nucleotide positions 5345 to 5397. Thisremoves the ATG start codon of S2 and the start codon of env. To makethe deletion within S2, PCR is carried out with SY2/SY5 and SY3/SY4using pONY2.13 DNA as template. The two PCR products are pooled and PCRis carried out with primers SY5 and SY3. The 10.1 kb product is ligatedinto pGEMT-easy (Promega) to make pGEMS2 and sequenced to confirm thedeletion. pONY2.13lacZDS2 is made by cutting out the 1.1 kb S2 regionfrom pGEMS2 with Cel II and ligating it into pONY2.13lacZ.

[0170] B) Gagpol Construct

[0171] The same region of S2 is deleted in pONY3 to preventrecombination between pONY3DS2 and pONY2.13DS2 reconstituting the S2gene. pONY3DS2 is made by PCR amplification with SY1/SY2 and SY3/SY4using pONY3 DNA as template. The two PCR products are pooled and PCR iscarried out with primers SY1 and SY3. The 0.7 kb product is ligated intopGEMT-easy (Promega) to make pGEMS22 and sequenced to confirm thedeletion. The 0.7 kb S2 coding region is excised of pGEMS22 with Not Iand inserted into pBluescript KS+(Stratagene) to make pBPCRS2. The EcoRV and Nco I fragment from pONY3 (2.2 kb) is inserted into pBPCRS2 cutwith Eco RV and Nco I to make pBpONYS2. This is then cut with Eco RV andCel II (2.9 kb fragment) and inserted into pONY3 cut with Eco RV and CelII to thereby making pONY3DS2.

[0172] Construction of dUTPase Mutant

[0173] pONY3DdUTPase is made by site directed mutagenesis of nucleotide4176 from a T to an A residue (Payne et al., Virology, 210:302-313).This mutates the aspartic acid to a glutamic acid. This is done by PCRamplification using PCR primers dUTPaseF and dUTPaseR. The template DNAis pONY³. The PCR product is inserted into pGEMT-easy and sequenced toconfirm the mutation. This is called pGDdUTPase. pONY3 is cut with Not Iand Eco RV (4.6 kb) and inserted into pBluescript KS+(Stratagene) tomake pBEV. The pGDdUTase is cut with Pac I and Pst I and the 0.4 kb bandinserted into pBEV cut with Pac I and Pst I. This is calledpONY3pBDUTPase. This is then inserted into pONY3 via N{overscore (o)}t Iand Eco RV (4.6 kb) to make pONY3DdUTPase.

[0174] Construction of the S2 and dUTPase Double Mutant

[0175] To make the double mutant of pONY3 the construct pBpONYS2 isused. pGDdUTPase is cut with Pac I and Pst I and the fragment insertedinto pBpONYS2 cut with Pac I and Pst I to make construct pS2DdUTPase.This is then cut with Eco RV and Cel II and inserted into pONY3 cut withEco RV and Cel II to make pONY3DS2DdUTPase.

[0176] Analysis of S2 and dUTPase Mutants

[0177] pONY2.13lacZDS2, pONY3DdUTPase and pONY3DS2DdUTPase vectors areused in a number of combinations in three plasmid co-transfections togenerate virus as described for MLV-based vectors (Soneoka et al 1995Nucl. Acids Res. 23:628-633) and the virus generated is titred on 293Tand D17 cells, in either dividing or non-dividing states. Cells arearrested in G₁/S phase by treatment with aphidicolin (9) and thentransduced with EIAV-based and MLV-based vectors pseudotyped with VSV-G(Table 4). The transduction efficiency of the MLV vector is lower byfour orders of magnitude in aphidicolin treated cells as compared tountreated cells. The incomplete block to cell transduction by MLV isprobably due to a small population of dividing cells. In contrast, nosignificant difference is observed in the case of the EIAV-basedvectors. This demonstrates that the EIAV-based system does not requireS2 or dUTPase either for production or transduction. Payne et al.,(Payne et al., Virology, 210:302-313) and others have shown that EIAVdUTPase is required for the infection of horse macrophages. This mayrepresent a restriction in infection of macrophages by EIAV.

[0178] The properties of the S2 and dUTPase mutants are tested bytransduction of hippocampal embryonic day 14 neuronal cells cultured inminimal media for 7 days. No significant difference is found between thevarious EIAV vectors. However a much reduced transduction efficiency isseen for the MLV vector. This indicates that S2 and dUTPase is notrequired for the transduction of physiologically non-dividing cells.

[0179] In summary we can conclude that tat, S2 and dUTPase are notrequired in any part of the vector system for vector production ortransduction.

EXAMPLE 8 Addition of Rev/RRE

[0180] The construction of pEGASUS-1 has been described above. Thisvector contains 759 bp of EIAV sequence. The introduction of the EIAVRRE (0.7 kb) into pEGASUS-1 to produce pEGASUS/RRE resulted in afour-fold increase in the titre when Rev is provided in trans (Table 2).This vector now contains 1.47 kb of EIAV.

Example 9 Construction of Improved Gagpol Expression Plasmids

[0181] In pONY3 there is an extended 5′ untranslated region before thestart of the gagpol coding sequence. It is likely that this unusuallylong sequence would compromise expression of the gagpol cassette. Toimprove gagpol expression pONY3 is modified to remove the remaining 5′LTR. This is done by cutting pONY3 with Nar I and Eco RV. The 2.4 kbfragment is inserted into pBluescript KS+(Stratagene) at Cla I and EcoRV sites to make construct pBSpONY3.0. pBSpONY3.0 is cut with Xho I andEco RV. The 2.4 kb fragment is inserted into pONY3 at Xho I and Eco RVto make pONY3.1.

[0182] This manipulation removes the 5′ LTR up to the Nar I site withinthe primer binding region (386 nt). This construct gives a two foldincrease in titre and increased protein expression (FIG. 10).

[0183] pONY3.1 like pONY3 encodes gag, gagpol, Tat, S2 and Rev. Sincethe S2 mutation experiments showed that S2 is not required either in theproduction system or in the EIAV vector genome it is possible to designa gagpol expression constructs without S2. Two such constructs, pHORSEand pHORSE3.1, are produced.

[0184] pHORSE is made by PCR amplification with EGAGP5′OUTERIEGAGPINNER3and EGAGP3′OUTER/EGAGPINNER5 using pONY3 as template DNA. The two PCRproducts are purified pooled and re-amplified using primersEGAGP5′OUTER/EGAGP3′OUTER. This product is inserted into the Xho I andSal I sites of pSP72 to make pSP72EIAVgagpolO'lap. pONY 3 is cut withPvu II and Nco I and the 4.3 kb fragment is inserted intopSP72EIAVgagpolO'lap cut with Pvu II and Nco I to make pSPEGP. This iscut with Xho I and Sal I (4.7 kb) and inserted into pCI-Neo at the Xho Iand Sal I sites. This construct is called pCIEGP. The RRE is cut outfrom pEGASUS with Sal I (0.7 kb) and inserted into pCIEGP construct atthe Sal I site to make pHORSE.

[0185] When this construct is assayed for protein expression in thepresence or absence of pCI-Rev (a construct expressing the EIAV Rev openreading frame, see above) it is found to be Rev dependent as expected.However, protein expression is much lower than from pONY3.1. In additionwhen used in virus production the titre is found to be 100 fold lowerthan that from pONY3.1.

[0186] Unexpectedly when the leader sequence (comprising sequences fromthe end of U5 of the 5′ LTR to the ATG start of gag 383-524 nt) ofpONY3.1 is inserted into pHORSE, to make pHORSE3.1, protein expressionand virus production improved. pHORSE3.1 is made by replacing the 1.5 kbXho IXba I of pHORSE with the 1.6 kb Xho I/Xba I of pONY3.1. Titresobtained with pHORSE3.1 are similar to that of pONY3.1. The reason forthe slightly lower titre of pHORSE3.1 compared to pONY3.1 may be due therequirement for a four plasmid co-transfection with pHORSE3.1 (due tothe Rev dependence of this system). We can conclude therefore that aminimal EIAV vector system should have this leader for maximum gagpolexpression.

[0187] When pHORSE3.1, pRV67, pCIRev and pEGASUS/RRE are used in a fourplasmid co-transfection (Table 6) virus is produced at a high titre(2.0×10⁴ l.f u./ml). This system lacks the second exon of Tat which isresponsible for Tat transactivation (Southgate et al., J. Virology,1995, 69:2605-2610). This demonstrates that the Tat is not required forthe EIAV-based vector system.

[0188] By engineering the backbone of pHORSE3.1 to express Rev(replacing the Neo open reading frame with that of EIAV Rev) therequirement of a four plasmid co-transfection was eliminated. This wasdone by cutting pCI-Neo with Stu I and Bst XI and filling in the 5′overhangs with T4 DNA polymerase. This produced a vector fragment of 4.6kb into which the Rev open reading frame from pTopoRevpos (cut with SacI and Xba I giving a 0.6 kb band in which the 5′ overhangs were filledin using T4 DNA polymerase) was inserted. This was called pCREV. TheEIAV gagpol reading frame including the RRE and leader was cut frompHORSE3.1 with Xho I and Not I (5.5 kb) and inserted into pCREV at theXho I and Not I sites to make pEGPR3.1.

[0189] Codon optimisation of the EIAV gagpol should eliminate thedependence of gagpol protein expression on the RRE/Rev system. The needof pEGASUS-1 for Rev/RRE can also be eliminated by using a heterologousRNA export system such as the constitutive transport element (CTE) fromMason-Pfizer Monkey virus (MPMV) (Bray et al., PNAS, 1994, 91:1256-1260,Kim et al., 1998) TABLE 4 Titre on Ratio D17 cells (Non- (l.f.u./ml)Non- dividing/ Vector gagpol Dividing dividing dividing) S2+ S2+,dUTPase+ 2.2 × 10⁵ 1.1 × 10⁵ 0.5 S2− S2+, dUTPase+ 1.5 × 10⁵ 1.3 × 10⁵0.9 S2− S2−, dUTPase+ 1.0 × 10⁵ 1.2 × 10⁵ 1.2 S2− S2−, dUTPase− 1.5 ×10⁵ 1.6 × 10⁵ 1.1 S2+ S2−, dUTPase+ 2.2 × 10⁵ 2.3 × 10⁵ 1.0 S2+ S2−,dUTPase− 1.5 × 10⁵ 1.4 × 10⁵ 1.0 S2+ S2+, dUTPase− 1.5 × 10⁵ 1.4 × 10⁵1.0 MLV Vector 1.2 × 10⁷ 6.7 × 10³ 0.0006 Mock <1 <1 1

[0190] TABLE 5 Comparison of pONY2.10LacZ and pEGASUS +/− EIAV RRE.Titre Vector Genome Gagpol (l.f.u./ml) pONY2.10LacZ pONY3.0   7 × 10⁴pEGASUS pONY3.0 2.2 × 10⁴ pEGASUS/RRE pONY3.0 8.6 × 10⁴

[0191] Titres with Rev are higher for pEGASUS-1 even though it has noRRE. Possibly the effect of REV is via enhanced expression of gagpol.TABLE 6 Titre Vector Genome Gagpol (l.f.u./ml) pONY2.11lacZ pONY3.1 1.7× 10⁵ pONY2.11lacZ pHORSE3.1 9.0 × 10⁴ pEGASUS/RRE pONY3.1 8.0 × 10⁴pEGASUS/RRE pHORSE3.1 2.0 × 10⁴

[0192] Transfections were carried out in 293T cells with pCI-Rev andpRV67. The virus was titred on D17 cells Primers

EXAMPLE 10 pONY4 Series of Vectors

[0193] In order to eliminate the use of Tat for the transcription of theEIAV genome and increase the amount of full length transcript the EIAVU3 (5′ LTR) was replaced with the HCMV enhancer/promoter as in the caseof the pEGASUS vectors (Example 2).

[0194] Plasmid Construction.

[0195] pONY2.1lacZ contains a deletion in gag such that only 373 bp ofthe gag ORF remains. pONY4 was made by replacing the 5′ LTR with the CMVLTR from pEGASUS-1. pEGASUS-1 was cut with Bgl II/Xho I releasing a 3.2kb fragment (containing the CMV LTR) which was inserted into pSP72 cutwith Bgl I/Xho L This construct was named pSPPEG213. This was cut withHpa I/Nar I and the 1.3 kb fragment (encompassing the CMV LTR) wasinserted into pONY2.11lacZ cut with Nae I/Nar L pONY4.1 contains adeletion (20.1 kb) downstream of the lacZ gene (between the Sfu I andSal I sites) such that tat, S2, env, rev and RRE, are either missing orseverely truncated (FIG. 11c). pONY4.1 was made by cutting it with SfiI/Sal I, blunt-ended by Klenow polymerase and religated. pONY4G was madeby replacing the lacZ gene of pONY4 (Sac II/Kpn I and then blunting withKlenow polymerase) with that of GFP from pEGFP-N1 (Clontech) (Bam HI/XbaI and then blunting with Klenow polymerase) as a blunt fragment.

[0196] Production and Assay of Vectors.

[0197] Vector stocks were generated by calcium-phosphate transfection ofhuman kidney 293T cells plated on 10 cm dishes with 16 μg of vectorplasmid, 16 μg of gag-pol plasmid and 8 ug of envelope plasmid. 36-48 hafter transfection, supernatants were filtered (0.45,um) aliquoted andstored at −70° C. Concentrated vector preparations were made byultracentrifugation of at 20 000 rpm (SW40Ti rotor) for 90 min, at 4° C.The virus was resuspended in PBS for 3-4 h aliquoted and stored at −70°C. Transduction was carried out in the presence of polybrene (8 μg/ml).It was consistently observed that pONY2.11 lacZ gave about 2 to 4 foldhigher titres than the less deleted pONY2.10lacZ. When U3 in the 5′ LTRwas replaced with the CMV enhancer/promoter as in pONY4 then titresincrease a further 5 to 10 fold.

EXAMPLE 11 EIAV ‘Self-Inactivating’ Vectors (SIN-Vectors)

[0198] The expression of the transgene from EIAV vectors in particularcell types may be influenced by elements in the LTR's. To remove suchelements SIN (Self Inactivating) vectors can be constructed however theprecise configuration of the vector may be influenced by the requirementto maintain certain sequences necessary for efficient production of thevector (Mol Cell Biol 1996 Sep; 16(9):4942-51. RNA structure is adeterminant of poly(A) site recognition by cleavage and polyadenylationspecificity factor. Graveley B R, Fleming E S, Gilmartin G M) (J Virol1996 Mar; 70(3):1612-7. A common mechanism for the enhancement of mRNA3′ processing by U3 sequences in two distantly related lentiviruses.Graveley B R, Gilmartin G M). In addition SIN vectors provide a way foreliminating the production of full length transcripts in transducedcells.

[0199] Two SIN vectors were made: one containing the putativelyimportant sequences (for polyadenylation), located between the Mlu I andMun I sites and one in which these sequences were deleted. The 5′ borderof the deletions was 112 bases from the 5′ end of the U3 region of the3′LTR. The structure of two SIN vectors is shown in FIG. 12.

[0200] Deletions present in pONY4G.SIN-MLU and pONY4G.SIN-MUN vectorsare indicated in dashed lines. Primers are shown in italic.

[0201] DNA sequences between nucleotides 7300 and 8079 (numberedaccording to EIAV clone pSPEAIV19, Accession No. U01866) were obtainedusing polymerase chain reaction amplification using pONY4G as template.The positive sense primer was ERRE3 and the negative primers foramplification were SIN-MLU (C7143: GTCGAGCACGCGTTTGCCTAGCAACATGAGCTAG(MluI site in bold) or SIN-MUN (C7142: GTCGAGCCAATTGTTGCCTAGCAACATGAGCTAG (MunI site in bold) where the underlined sequences arecomplimentary to nucleotides 8058 to 8079 (of pSPEIAV19). The PCRproducts were digested with NspV and either MuI or MunI respectively.These were then ligated into pONY4G prepared for ligation by digestionwith NspV(SfuI) and either MluI (partial digestion) or MunIrespectively.

EXAMPLE 12 EIAV Vectors with Reverse Configuration InternalPromoter-Reporter Cassettes

[0202] In EIAV vectors such as pONY4Z or pONY4G the internalCMV-reporter cassette is orientated so that transcription from the 5′LTRand the internal promoter are co-directional and the polyadenylationsignal in the 3′LTR is used for transcripts from both promoters. Analternative configuration is achieved by reversing the internalpromoter-reporter cassette, however a polyadenylation signal must beplaced downstream of the cassette.

[0203] An example of this ‘reverse orientation’ vector was made asfollows. pONY4Z was digested with PstI and the overhanging terminitrimmed back with T4 DNA polymerase. This was then used as the ‘vector’fragment in a ligation with the MluI to AseI fragment from pEGFP-C1which contains sequences including the CMV-GFP-SV40 early mRNA polyAsignal cassette. Prior to ligation this fragment was flush-ended with T4DNA polymerase. The vector encoding plasmid was called pONY4Greverse.

[0204] Vector particles were recovered from pONY4Greverse byco-transfection with pONY3.1 and pRV67, which express EIAV gag/pol andVSV-G protein respectively. The titre on D17 canine cells frompONY4Greverse was 13-fold lower than from pONY4G vector recovered inparallel.

[0205] The lower titre of pONY4Greverse was probably due to interferencebetween the CMV promoters which drive transcription of the genome andthe GFP towards each other however truncation of the genomic RNA by theSV40-derived polyadenylation signal present in the insertedCMV-GFP-polyA cassette could also have been a factor. An improved vectorwas made by replacing the polyadenylation signal of pONY4Greverse withthe bovine growth hormone polyadenylation (BGHpA) signal. To make thisimprovement pONY4Greverse was digested with BstAPI and the ends flushedwith T4 DNA polymerase, then cut with PstI. This ‘vector’ fragment wasthen ligated to a DNA fragment representing the BGHpA which was preparedfrom pcDNA3.1+ (Invitrogen) by digestion with SphI, and then the endsblunted with T4 DNA polymerase, then digested with PstI.

EXAMPLE 13 Construction and use of poly.A Signals Containing Introns

[0206] In the pONY vectors described here the polyadenylation signalused is that from EIAV. This is found in the 3′ LTR at the border of Rand U5. This signal may not be optimal because it is not of a consensussequence (see Whitelaw and Proudfoot 1986 EMBO 5; 2915-2922 and Levittet al 1989 Gen. and Dev. 3; 1019-1025 for description of consensuspolyadenylation signal).

[0207] One method of improving the viral polyadenylation is to replacethe 3′ LTR poly A signal with that of a consensus/strong polyadenylationsignal. By such a method the signal would now be optimal in the producercell. However upon transduction this signal is lost because duringreplication, the 5′ LTR is the source of the poly A signal (seeRetroviruses 1998 CSH press (Ed. J. Coffin) for review of retrovirallife cycle). One novel way of overcoming the problem (of no strongpolyadenylation signal upon transduction) is to include the poly Asignal in a manner as will now be outlined: The method is to use a‘split poly-A signal’ where by an intron splits the aataaa motif fromthat of the essential g/u box. Such a signal has previously been used byLiu et al (1993 N.A.R 21;5256-5263) to demonstrate both that large gapsbetween the aataaa and the g/u box will disable the poly A signal andthat the polyadenylation process preceeds splicing. By placing asplit-polyA signal within the retroviral vector such a signal will notbe functional until transduction of target cells. This is becausepolyadenylation preceeds splicing and as such the upstream split-polyAsignal will not be used during vector expression within the producercell. Outlined in FIG. 13 is a schematic representation of how such aretroviral vector, containing a split polyA signal, would function—bothin producer and in transduced cells. First this Figure demonstrates thatalthough there exists an upstream consensus polyadenylation signal, theinitial vector transcripts are still polyadenylated at the usual 3′ LTRusing either a viral or other poly A signal as so desired. This isbecause although the upstream poly A signal is functional in the finalvector genome, this signal is not read by the polyadenylation machinerybecause it is created only during intron removal and thus not present inthe primary RNA transcript. Second, this figure demonstrates that upontransduction the-resulting vector transcripts are now polyadenylated atthe first signal; this being now a normal strong polyadenylation signalwith no introns to distance the essential aataaa and g/u box.

[0208] There are a number of advantages to inclusion of such asplit-poly A signal within a retroviral vector; these include thefollowing:

[0209] (1) The use of strong non-viral based polyadenylation signalwithin the transduced cell will enhance gene expression within suchcells.

[0210] (2) The use of such poly A signals upstream of the natural LTR(see FIG. 13) based signals will, upon transduction, generate shorterRNA transcripts that contain less viral sequence at their 3′ end and assuch will not be able to undergo subsequent retroviral reversetranscription. Indeed if the desired gene is expressed from an internalpromoter such as the CMV, rather than an LTR; the resulting transcriptexpressed in the transduced cell could be designed to contain no viralsequence at all (see FIG. 3A).

[0211] (3) Inclusion of such a signal upstream of the 3′LTR will meanexpression of the RNA downstream to the split poly A signal will belimited only to the producer cell because such RNA will not betranscribed in the transduced cell. This will therefore restrict certainsequence expression (for example IRESneo; see FIG. 14B) to producercells.

[0212] (4) The presence of an intron within the producer cell will helpwith nuclear export of vector RNA from the nucleus.

[0213] (5) Because upon transduction their now exists an internalfunctional poly A signal, the viral poly A signal in the 5′ LTR (the onecopied to the 3′ position during reverse transcription) can beremoved/deleted if desired. This is of use for preventing the process ofpromoter-proximal polydenylation from the 5′ LTR in the producer cell(see Scott and Imperiale 1997 (Mol. Cell. Biol. 17;2127-35) and thusencourage full length transcript production of the virus.

EXAMPLE

[0214] To demonstrate the use of such a signal in a retrovirus; the“split poly A signal” cassette is constructed as described in FIG. 15;with the intronic sequence being derived from pCI (Promega). Once madethis cassette is cloned into the pONY 4 GFP vector using the PstIcompatible unique sse8387 site of pONY4-GFP (see FIG. 16). Upontransduction the resulting vector will now polyadenylate prior to the3′LTR and consequently no viral RNA 3′ to lacZ will be transcribed (seeFIG. 16).

EXAMPLE 14 Construction of MLV/EIAV Vectors

[0215] By replacing the EIAV LTR sequences with the MLV equivalents, thepONY vectors will no longer possess functional tar elements within therepeat regions (R) and as a consequence the U3 promoter will functionwithout the requirement of Tat in the transduced cell.

[0216] Outlined in FIG. 17 is how such a vector is made by overlappingPCR with primers described in FIG. 18. Primers Mel and Me2 are used toamplify a PCR product from the MLV vector pHIT111 (Soneoka et al 1995NAR 23;628-633) whilst Me3 and Me4 are used to amplify a product frompONY4 lacZ. The resulting two products are then combined in a primerlessPCR reaction to overlap them (homology between the two products isshaded in FIG. 17). The final full length product is cut BglII and Xbaland used to replace the BglII-Xbal fragment of pONY4 lacZ (containingthe CMV/R/U5) to make pONY4-5′MLV. The resulting vector now has theCMV/R/U5 sequence from MLV linked to the EIAV U5 sequence (sequencerequired for genome recognition by intergrase prior to intergration).The next step involves PCR amplification with primers Me5 and Me6 frompONY4 LacZ template and PCR amplification with Me7 and Me8 from pLXSNtemplate (Miller and Rosman 1989 Biotechniques 7:980-990). These two PCRproducts are then overlapped by primerless PCR (homology between primersshown as hatched box) and the resulting fragment cut with NspV and MunIand inserted into the NspV/MunI sites of pONY4-5′MLV; thus replacing the3′EIAV LTR with a 3′MLV LTR fused to the 3′UTR/ppt/U3 intergrase bindingsite of pONY 4 lacZ. The resulting plasmid, named pONY-MOUSE (see FIG.19 for complete DNA sequence), titres at 10⁴⁻⁵ per ml when combined withpONY3.1 and pRV67 in the HIT system.

EXAMPLE 15 Early Promoter Driving Lentiviral Vector Genome

[0217] In this example an EIAV genome is expressed from a vaccinia earlypromoter P7.5E (Davison 1989a). The promoter has been engineered toproduce an EIAV genome with the correct 5′ RNA end. In addition thevaccinia early termination sequence has been inserted downstream of theEIAV genome. This is inserted into the transfer plasmid pSC65, which canhomologously recombine into the TK region of the MVA genome. Recombinantviruses can be selected by their lack of sensitivity to BudR (Earl etal. 1998).

[0218]FIG. 11 is a schematic representation of the EIAV genome vectorspONY4.0 and pONY4.1 which have been described in Example 10 and thevaccinia transfer vector pSC65 (Chakrabarti et al 1997). The P7.5Esequence is AAAAGTAGAAAATATATTCTAATTTATT. The Early termination sequencefor the early promoters is TTTTTNT (N=any nucleotide) (Fields).

[0219] The DNA manipulations are as follows and FIGS. 20 and 21 give thesequence of the PCR primers. PCR with primers EMVA1/2 produces the 5′LTR with the U3 region replaced by the P7.5E promoter. This is insertedinto the plasmid pSP72 (Promega) using the Hind III/Pst I sites to makepEMVA1. EIAV U3 contains a sequence matching the criteria for vacciniaearly termination (TTTTTAT). Using primers EMVA3/4 and EMVA5/6 andoverlapping PCR this region is mutated to TTTCCAT in order to preventearly termination. This PCR product is inserted into the pEMVA1 usingthe Bgl II/Pst I sites to generate pEMVA2. A termination sequence(TTTTTTTTT) is inserted downstream of the 3′ LTR R region using primersEMVA7/8 . This PCR product is inserted into pEMVA2 using the Mun I/BglII sites making pEMVA3. Into this plasmid the rest of the EIAV vectorgenome (pONY4) is inserted via the Nar I/Nsp V sites making pEMVA4 (FIG.22). This is then cut with Pac I/Bgl II and inserted into pSC65 cut withPac I/Bam HI to make pEPONY4 (Bgl II and Bam HI are compatible) (FIG.23). This removes the two vaccinia promoters and the lacZ codingcassette from pSC65.

[0220] In order to make the minimal EIAV genome version of thisconstruct that is analogous to pONY4.1, pEMVA4 is cut with Sal I/Nsp Vblunt ended and religated to make pEMVA5 (FIG. 24). This removes much ofthe sequence between the end of the lac Z gene and the end enveloperegion, hence this vector is Tat, Rev, S2 and Env minus. This isdescribed in Example 10. This is then cut with Pac I/Bgl II and insertedinto pSC65 cut with Pac I/Bam HI to make pEPONY4.1 (Bgl II and Bam HIare compatible) (FIG. 24). This removes the two vaccinia promoters andthe lacZ coding cassette from pSC65.

[0221] Both pEPONY4.0 and pEPONY4.1 are suitable for inserting thegenome expression cassettes into the TK region of the MVA genome(Carroll MW and Moss B Virology Nov. 24, 1997;238(2):198-211) using aBHK TK-ve cell line (ECACC 85011423) and standard procedures for theconstruction of recombinant poxviruses (Earl et al 1998a & 1998b)

EXAMPLE 16 Synthetic Early/Late Promoter Driving Lentiviral VectorGenome

[0222] The synthetic early/late promoter of vaccinia has a requirementfor sequences downstream of the RNA initiation site (Davison 1989b). Forthis promoter to be used to generate a retroviral genome either the Rregions have to be modified or a ribozyme is used to make the correct 5′end. Modifying the R regions is problematic as the initiation site hasnot been conclusively identified and varies with the sequence (Davison1989b). Below is described the generation of a transfer plasmid thatexpresses the EIAV genome from the synthetic early/late promoter (Psyn).Downstream of this promoter is inserted a ribozyme that ensures thecreation of the correct 5′ end of the RNA. This construct also containsthe early termination sequence.

[0223] The DNA manipulations are as follows: PCR with primers EMVA9/1produces the 5′ LTR with the U3 region replaced by the Psyn promoter anda hammerhead ribozyme (FIGS. 25 and 26). This is inserted into theplasmid pEMVA4 (Example 15) using the Pac I/Nar I sites to make pEMVA6(FIG. 27). This is then cut with Pac I/Bgl II and inserted into pSC65cut with Pac I/Bam HI to make pSynPONY4 (Bgl II and Bam HI arecompatible) (FIG. 27). This removes the two vaccinia promoters and thelacZ coding cassette from pSC65. In order to make the minimal ELAVgenome version of this construct that is analogous to pONY4.1, pEMVA6 iscut with Sal I/Nsp V blunt ended and religated to make pEMVA7 (FIG. 28).This is then cut with Pac I/Bgl 11 and inserted into pSC65 (a vacciniatransfer vector) cut with Pac I/Bam HI to make pSynPONY4.1 (Bgl II andBam HI are compatible) (FIG. 28). This removes the two vacciniapromoters and the lacZ coding cassette from pSC65.

[0224] Both pSynPONY4.0 and pSynPONY4.1 are suitable for inserting thegenome expression cassettes into the TK region of the MVA genome(Carroll MW and Moss B Virology Nov. 24, 1997;238(2):198-21 1) usingstandard procedures for the construction of recombinant poxviruses (Earlet al 1998a & 1998b)

EXAMPLE 17 T7 Promoter Driving Lentiviral Vector Genome

[0225] The T7 promoter can be used to generate a retroviral genome whichcan make the correct 5′ end. Below is described the generation of atransfer plasmid that expresses the EIAV genome from the T7 promoter(T7). Downstream of this promoter is inserted a T7 termination sequence.This is inserted into the transfer plasmid pSC65, which can homologouslyrecombine into the TK region of the MVA genome. The T7 promoter requiresthe T7 polymerase. MVA viruses are available which express T7 polymerasefrom Vaccinia promoters (Wyatt et al 1995).

[0226] The T7 promoter has the sequence (−)TAATACGACTCACTATAGG(+2) withtranscription beginning after A with preferably a run of Gs. The T7termination sequence isCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG. The T7 promoter andterminator sequences are as those described in the plasmid pCITE-4a(+)(Novagen).

[0227] The DNA manipulations are as follows. PCR with primers EMVA10/11(FIG. 29) produces the 5′ LTR with the U3 region replaced by the T7promoter. This is inserted into the plasmid pEMVA4 using the Pac liNar Isites to make pEMVA8 (FIG. 30). PCR with primers EMVA 1/7 produces partof the 3′ LTR with a T7 termination sequence. This is inserted intopEMVA8 using the Mun I/Bgl II sites to make pEMVA9. This is then cutwith Pac I/Bgl II and inserted into pSC65 (a vaccinia transfer vector)cut with Pac I/Bam HI to make pT7PONY4 (Bgl II and Bam HI arecompatible) (FIG. 30). This removes the two vaccinia promoters and thelacZ coding cassette from pSC65.

[0228] To make the minimal EIAV genome version of this construct(pONY4.1) pEMVA9 is cut with Sal I/Nsp V blunt ended and religated tomake pEMVA10 (FIG. 31). This is then cut with Pac I/Bgl II and insertedinto pSC65 (a vaccinia transfer vector) cut with Pac I/Bam HI to makepT7PONY4.1 (Bgl II and Bam HI are compatible) (FIG. 31). This removesthe two vaccinia promoters and the lacZ coding cassette from pSC65.

[0229] Both pT7PONY4.0 and pT7PONY4.1 are suitable for inserting thegenome expression cassettes into into the TK region of the MVA genome(Carroll MW and Moss B Virology 1997) using standard procedures for theconstruction of recombinant poxviruses (Earl et al 1998a & 1998b).

EXAMPLE 18 Construction of an EIAV Gagpol Cassette for Expression inVaccinia

[0230] Normally EIAV gag/pol requires Rev/RRE for expression as Revenables the unspliced transcript to be exported out of the nucleus. AsPox viruses are cytoplasmic, EIAV viral RNA export should not be aproblem. But if Rev has other functions such as RNA stability or acts asa translation enhancer it can be expressed in a similar way to EIAVgag/pol (Martarano 1994). Alternatively the EIAV gag/pol sequence can becodon optimised to overcome the Rev/RRE requirement for export andenhance RNA stability. Below is described the creation of a vector thatexpresses EIAV gag/pol from a synthetic early/late promoter (Psyn). Thisis inserted into the transfer plasmid pLW-22 (Wyatt and Moss Appendix 1,Earl et al 1998a & b), which can homologously recombine into the Del IIregion of the MVA genome. Recombinant viruses can be selected by theirexpression of lac Z.

[0231] EIAV gag/pol including the leader the gag/pol open reading frameand the RRE can be obtained from cutting pHORSE3.1 (Example 9) with AhoLINot I to give a 5.5 kb band (FIG. 32). This is then inserted into thevaccinia transfer vector pLW-22 cut with Sal I/Not I (Sal I and Xho Iare compatible) to make pLWHORSE3.1 (FIG. 32).

EXAMPLE 19 Construction of an EIAV Rev Cassette for Expression inVaccinia

[0232] In the event that Rev is required for EIAV viral vectorproduction from a poxvirus it can be expressed from a syntheticearly/late promoter. This construct is inserted into the transferplasmid pMC03, which can homologously recombine into the Del III regionof the MVA genome. Recombinant viruses can be selected by theirexpression of GUS (Carroll et al. 1995).

[0233] The DNA manipulations are as follows. Plasmid pCIRev is describedin Example 9. It is an EIAV Rev expression plasmid. This is cut with AflII/Not I (0.6 kb), blunt ended by T4 DNA polymerase and inserted intopMC03 (Carroll et al. 1995) cut with Pme I to make pMCRev (FIG. 33).

EXAMPLE 20 Construction of Heterologous Envelope Cassettes forExpression in Vaccinia

[0234] EIAV can be pseudotyped with a number of envelopes such as VSV-Gand amphotropic MLV envelope. Below is described the creation of a MVAtransfer vector that expresses the amphotropic envelope or VSV-Genvelope from the P7.5 early/late promoter. The transfer vector is pYF6which can homologously recombine into the HA region of MVA. Recombinantviruses can be selected by direct live immunostaining for expression ofthe env.

[0235] In order to produce a transfer vector containing a VSV-Gcassette, the VSV-G expression plasmid pRV67 (Kim et al. 1998) is cutwith Sma I/Eco RV(1.7 kb) and the resulting fragment inserted into pYF6cut with Sma I to make pYFVSVG (FIG. 34). Similarly, to produce ananalogous amphotropic envelope construct pHIT456 (Soneoka 1995) is cutwith Xba I and the 2.2 kb band blunt ended by T4 DNA polymerase andinserted into pYF6 cut with Sma Imaking pYFAmpho (FIG. 35).

[0236] Both pYFAmpho and pYFVSVG are suitable for inserting the genomeexpression cassettes into into the HA region of the MVA genome usingstandard procedures for the construction of recombinant poxviruses (Earlet al 1998a & b, Flexner et al 1987)

EXAMPLE 21 Construction and Amplification of MVA-Lenti Recombinants

[0237] The recombinant vaccinia viruses containing multiple insertsencoding the components of the EIAV vectors (FIG. 25) are constructed bysequential recombination with the relevant transfer plasmids. Theconstruction of v.MEeG-Or (FIG. 36) is used as an example:

[0238] 1. A plasmid carrying gag-pol (pLWHORSE3.1) is transfected intoBHK-21 or CEF cells, that have been previously infected with MVA (asdescribed in Carroll and Moss 1997, Earl et al 1998a & b).

[0239] 2. After two days of infection recombinant MVA virus is assayedon BHK-21/CEF and cells are over-layed with agar medium containing thesubstrate for the colour marker P-galactosidase (Chakrabarti et al 1985)which is expressed from within pLW22.

[0240] 3. Blue plaques are picked and plaque purified until ahomogeneous recombinant virus population is obtained.

[0241] 4. Recombinant virus is then used to recombine with transferplasmids containing the other recombinant genes: pMCRev in whichselection is based on GUS expression (Carroll & Moss 1995), the genome(pEPONY4.0) in which selection is based on a TK negative phenotype usingBudR (Carroll & Moss 1997, Earl et al 1998a & b) and VSVG (PYFVSVG) inwhich recombinants are identified by direct immunostaining of VSV G(Earl et al 1998a & b).

[0242] Recombinant viruses may be amplified in BHK-21 or CEF cells asdescribed below:

[0243] Propagation of Vaccinia Virus

[0244] The highly attenuated strain MVA is derived from the replicationcompetent strain Ankara and has endured over 570 passages in primarychick embryo fibroblast cells. MVA replication was initially thought tobe restricted to CEF cells as only minimal replication in mammaliancells was reported. However, further analysis has shown that BabyHamster Kidney cells (BHK-21) are able to support high titre productionof MVA. MVA may thus be grown on BHK-21 or primary CEF cells (Carroll &Moss (1997) Virology 238:198-211).

[0245] To prepare CEF cells, 10 day old chick embryos are gutted andlimbs and head are removed before being minced and trypsinised in asolution of 0.25% trypsin and incubation at 37° C. The cell suspensionis filtered through a course filter mesh and cells are washed andconcentrated by centrifugation at 2000 rpm in a Sorvall RC-3B at 1500rpm for 5 mins. Cells are suspended in MEM containing 10% FCS,aliquotted into 175 cm flasks and incubated at 37° C. in a CO₂incubator. When monolayers are 95% confluent they are trypsinised andused to seed additional flasks or six well plates. Alternatively,primary cultures are transferred to a 31° C. incubator for later use(Sutter and Moss (1992) Proc Natl Acad Sci U S A 89:10847-10851).

[0246] Preparation of Crude, Semi-Purified and Purified Virus Stocks

[0247] Crude virus stocks are prepared for initial recombinant virusanalysis or as viral stocks used for subsequent high titre viruspreparations. Vaccinia virus preparations can be semi-purified bycentrifuging out cell membranes and nuclei or by additional stepsinvolving sucrose centrifugation to prevent contamination bypre-expressed recombinant protein products and cellular organelles.Methods used are a modification of those described by Earl et al., 1998a& b; Earl and Moss, ibid, pp. 16.17.1-16.17.16; Earl and Moss, ibid, pp.16.18.1-16.18.10; and Bronte et al., (1997) Proc Natl Acad Sci U S A94(7):3183-3188.

[0248] Crude Virus

[0249] MVA is grown in either CEF or BHK-21 (obtained from the ATCC) andWR is grown in HeLa or BS-C-1 (ATCC) in 175 cm² tissue culture flasks.Briefly, confluent monolayers are infected with an moi of approx. 1 pfuwith MVA or WR. Virus is suspended in 10 ml MEM containing 2% FCS andadded to 175 cm² flasks containing confluent cell monolayers. Afterinoculation for 1 hour at 37° C. an additional 20 ml MEM containing 2%FCS is added. After 48-72 hours infected cells are scraped into themedium and pelleted at 1500 g for 5 mins. For crude virus preparationscells are resuspended 2 ml MEM (2%) per 175 cm² flask. Cells are freezethawed three times, sonicated and aliquotted into 1 ml freezing tubes. Arepresentative aliquot is freeze thawed and titred to determine virusconcentration. Virus stocks are stored below −20° C.

[0250] Semi-Pure Preparations

[0251] Infected cells are harvested as described previously (Earl et ala & b; Earl and Moss; 1991). After centrifugation cells are resuspendedin PBS (2 ml/175 cm² flask) and homogenised by 30-40 strokes in a tightfitting glass dounce homogeniser, on ice. Cell breakage is checked bymicroscopy. Nuclei, cellular organelles and membranes are removed by acentrifugation at 300 g for 5 mins (4° C.), keep supernatant. The cellpellet is resuspended in 1 ml/175 cm² flask and centrifugation repeated.The supernatants are pooled, aliquoted and stored.

[0252] Purified Preparation

[0253] Infected cells are harvested as previously described (Earl etal.a & b; Earl and Moss; 1991) and resuspended in 10 mM Tris.Cl, pH 9.0(2 ml/flask), keeping samples on ice from this point of the procedure.Homogenise as described previously using 10 mM Tris. The lysate issonicated (on ice) using an XL 2015 sonicating cup (Misonics, USA) atmaximum output (500 W) for 1 min. The sample is placed on ice for 1 minand the sonication repeated up to 3 times. A maximum of 5 ml issonicated at a time, and ice is replenished during sonication. Thelysate is gently layered onto a cushion of 17 ml of 36% sucrose (in 10mM Tris.Cl, pH 9.0) in a SW-27 centrifuge tube. Lyates are centrifugedfor 80 mins in an SW-27 rotor at 13 500 rpm (32,900×g), 4° C. Thesupernatant is discarded and the viral pellet resuspended in sterile PBSand sonicated in a cup sonicator for 1 min (on ice). Concentrated virusis aliquoted and stored at below −20° C.

EXAMPLE 22 Production of EIAV Vector Particles from MVA-EIAV Hybrids

[0254] As described above large scale preparations of recombinantMVA-EIAV are made. These preparations are used to infect mammalian cellsthat are non-permissive for MVA, such that the resulting supernatantwill only contain EIAV and not infectious MVA (Meyer et al 1991, Carrolland Moss 1997). A suitable cell line is MRC5 (ATCC). Cells are infectedat an MOI of 3. Infections are allowed to run for approximately 48 hoursbefore supernatants are harvested and EIAV vector particles either useddirectly or concentrated/purified by ultracentrifugation or cross-flowmethods. To produce large scale preparations, are grown in suspension oron microcarriers or in roller bottles. EIAV vectors carrying gene ofinterest prepared in these ways are used to transduce target cells invivo or in vitro.

REFERENCES

[0255] Blomer, U., Naldini, L., Kafri, T., Trono, D., Verma, I. M., andGage, F. H. (1997). Highly efficient and sustained gene transfer inadult neurons with a lentivirus vector. J Virol 71, 6641-6649.

[0256] Blomer, U., Naldini, L., Verma, I. M., Trono, D., and Gage, F. H.(1996). Applications of gene therapy to the CNS. Hum Mol Genet 5 SpecNo, 1397-404.

[0257] Clever, J., Sassetti, C., and Parslow, T. G. (1995). RNAsecondary structure and binding sites for gag gene products in the 5′packaging signal of human immunodeficiency virus type 1. J Virol 69,2101-9.

[0258] Clever, J. L., and Parslow, T. G. (1997). Mutant humanimmunodeficiency virus type 1 genomes with defects in RNA dimerizationor encapsidation. J Virol 71, 3407-14.

[0259] Fields, B. N., Knipe, D. M., and Howley, P. M. (1996). FieldsVirology, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman and S.E. Straus, eds. (Philadelphia. New York: Lippincott—Raven Publishers).

[0260] Fuller S, von Bonsdorff CH, Simons K. Vesicular stomatitis virusinfects and matures only through the basolateral surface of thepolarized epithelial cell line, MDCK. Cell 1984 Aug;38(1):65-77

[0261] Harrison, G. S., Long, C. J., Maxwell, F., Glode, L. M., andMaxwell, I. H. (1992). Inhibition of HIV production in cells containingan integrated, HIV-regulated diphtheria toxin A chain Gene. AIDsResearch and Human Retrovirus 8, 39-45.

[0262] Hayashi T, Shioda T, Iwakura Y, Shibuta H. RNA packaging signalof human immunodeficiency virus type 1. Virology 1992June;188(2):590-599

[0263] Kim V. N., Mitrophanous K., Kingsman S. M., Kingsman A. J. 1998.Minimal Requirement for a Lentiviral Vector Based on HumanImmunodeficiency Virus Type 1. J. Virol. 1998 72:811-6.

[0264] Kim, S. Y., R. Byrn, J. Groopman, and D. Baltimore. 1989.Temporal aspects of DNA and RNA synthesis during human immunodeficiencyvirus infection: evidence for differential gene expression. J. Virol.63:3708-3713.

[0265] Lewis, P. F., and M. Emerman. 1994. Passage through mitosis isrequired for oncoretroviruses but-not for the human immunodeficiencyvirus. J Virol. 68:510-6.

[0266] Mann R, Mulligan RC, Baltimore D. Construction of a retroviruspackaging mutant and its use to produce helper-free defectiveretrovirus. Cell 1983 May;33(1):153-159

[0267] Martarano, L., Stephens, R., Rice, N., and Derse, D. (1994).Equine infectious anemia virus trans-regulatory protein Rev controlsviral mRNA stability, accumulation, and alternative splicing. J Virol68, 3102-11.

[0268] Naldini, L., Blomer, U., Gage, F. H., Trono, D., and Verma, I. M.(1996). Efficient transfer, integration, and sustained long-termexpression of the transgene in adult rat brains injected with alentiviral vector. Proc Natl Acad Sci U S A 93, 11382-11388.

[0269] Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage,F. H., Verma, I. M., and Trono, D (1996). In vivo gene delivery andstable transduction of nondividing cells by a lentiviral vector [seecomments]. Science 272, 263-7.

[0270] Payne, S. L., Rausch, J., Rushlow, K., Montelaro, R. C., Issel,C., Flaherty, M., Perry, S., Sellon, D., and Fuller, F. (1994).Characterization of infectious molecular clones of equine infectiousanaemia virus. J Gen Virol 75, 425-9.

[0271] Yee, J.-K., M. Atsushi, P. LaPorte, K. Bouic, J. C. Bums, and T.Friedmann (1994) A general method for th generation of high-titer,pantropic retroviral vectors: Highly efficient infection of primaryhepatocytes. Proc. Natl. Acad. Sci. USA 91:9564-9568.

[0272] Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D.(1997). Multiply attenuated lentiviral vector achieves efficient genedelivery in vivo. Nat Biotechnol 15, 871-875.

[0273] Cannon 1996 J Virol 1996 70:8234-40. Murine leukemia virus-basedTat-inducible long terminal repeat replacement vectors: a new system foranti-human immunodeficiency virus gene therapy. Cannon P M, Kim N,Kingsman S M, Kingsman A J.

[0274] Carroll M W, Moss B E. coli beta-glucuronidase (GUS) as a markerfor recombinant vaccinia viruses. Biotechniques 1995 19:352-4

[0275] Carroll MW, Moss B Host range and cytopathogenicity of the highlyattenuated MVA strain of vaccinia virus: propagation and generation ofrecombinant viruses in a non-human mammalian cell line. Virology 1997238:198-211

[0276] Chakrabarti S, Brechling K, Moss B Vaccinia virus expressionvector: coexpression of beta-galactosidase provides visual screening ofrecombinant virus plaques. Mol Cell Biol 1985 12:3403-9

[0277] Chakrabarti S, Sisler J R, Moss B Biotechniques 1997 6:1094-7Compact, synthetic, vaccinia virus early/late promoter for proteinexpression. Davison 1989a J Mol Biol 1989 20;210(4):749-69. Structure ofvaccinia virus early promoters. Davison A J, Moss B

[0278] Davison 1989b J Mol Biol 1989 210(4):771-84. Structure ofvaccinia virus late promoters. Davison A J, Moss B

[0279] P L. Earl (a), N. Cooper, L S Wyatt, B. Moss & M. W. Carroll 1998Preparation of Cell Cultures and Vaccinia Virus Stocks. CurrentProtocols in Molecular Biology Supplement 43 Unit 16.16. John Wiley andSons Inc.

[0280] P L. Earl (b), B. Moss L. S. Wyatt, & M. W. Carroll 1998Generation of Recombinant Vaccinia Viruses. Current Protocols inMolecular Biology. Supplement 43 Unit 16.17. Current Protocols inMolecular Biology. John Wiley and Sons Inc.

[0281] Flexner C, Hugin A, Moss B Nature 1987 330(6145):259-62Prevention of vaccinia virus infection in immunodeficient mice byvector-directed IL-2 expression.

[0282] Holzer G W, Falkner F G Construction of a vaccinia virusdeficient in the essential DNA repair enzyme uracil DNA glycosylase by acomplementing cell line. J Virol 1997 71:4997-5002

[0283] Kim V N, Mitrophanous K, Kingsman S M, Kingsman A J Minimalrequirement for a lentivirus vector based on human immunodeficiencyvirus type 1. J Virol 1998 72:811-6

[0284] Mackett M, Smith G L, Moss B Vaccinia virus: a selectableeukaryotic cloning and expression vector. Proc Natl Acad Sci U S A 19827923:7415-9

[0285] Mahnel H, Mayr A Berl Munch Tierarztl Wochenschr 1994Aug;107(8):253-6 [Experiences with immunization against orthopox virusesof humans and animals using vaccine strain MVA].[Article in German]Zentralbl Bakteriol [B] 1978 Dec; 167(5-6):375-90

[0286] Martarano L, Stephens R, Rice N, Derse D Equine infectious anemiavirus trans-regulatory protein Rev controls viral mRNA stability,accumulation, and alternative splicing. J Virol 1994 May;68(5):3102-11

[0287] Mayr A, Stickl H, Muller H K, Danner K, Singer H [The smallpoxvaccination strain MVA: marker, genetic structure, experience gainedwith the parenteral vaccination and behavior in organisms with adebilitated defence mechanism]. Zentralbl Bakteriol [B]. 1978Dec;167(5-6):375-90. German.

[0288] Meyer H, Sutter G, Mayr A Mapping of deletions in the genome ofthe highly attenuated vaccinia virus MVA and their influence onvirulence. J Gen Virol 1991 72:1031-8

[0289] Moss B Poxviridae: The viruses and their replication Chapter 83.p2637-2672. In Fields, B. N., Knipe, D. M. & Howley, P.M. FieldsVirology. Third Edition edn Vol. 2 eds Chanock, R. M., Melnick, J. L.,Monath, T. P., Roizman, B. & Straus, S. E. Lippincott—Raven Publishers,Philadelphia. New York, 1996

[0290] Moss B, Carroll M W, Wyatt L S, Bennink J R, Hirsch V M,Goldstein S, Elkins W R, Fuerst T R, Lifson J D, Piatak M, Restifo N P,Overwijk W, Chamberlain R, Rosenberg S A, Sutter G Host rangerestricted, non-replicating vaccinia virus vectors as vaccinecandidates. Adv Exp Med Biol 1996;397:7-13

[0291] Panicali D, Paoletti E Construction of poxviruses as cloningvectors: insertion of the thymidine kinase gene from herpes simplexvirus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci U SA 1982 79:4927-31

[0292] Soneoka Y, Cannon P M, Ramsdale E E, Griffiths J C, Romano G,Kingsman SM, Kingsman A J 1995 Nucleic Acids Res 1995 23628-33. Atransient three-plasmid expression system for the production of hightiter retroviral vectors.

[0293] Sutter G, Moss B Nonreplicating vaccinia vector efficientlyexpresses recombinant genes. Proc Natl Acad Sci U S A Nov. 15,1992;89(22):10847-51

[0294] Taylor J, Weinberg R, Tartaglia J, Richardson C, Alkhatib G,Briedis D, Appel M, Norton E, Paoletti E Nonreplicating viral vectors aspotential vaccines: recombinant canarypox virus expressing measles virusfusion (F) and hemagglutinin (HA) glycoproteins. Virology 1992Mar;187(1):321-8

[0295] Paoletti E, Tartaglia J, Taylor J Safe and effective poxvirusvectors—NYVAC and ALVAC. Dev Biol Stand 1994;82:65-9

[0296] Wyatt L S, Moss B, Rozenblatt S Replication-deficient vacciniavirus encoding bacteriophage T7 RNA polymerase for transient geneexpression in mammalian cells. Virology Jun. 20, 1995;210(1):202-5

[0297] Wyatt L S, Carroll M W, Czerny C P, Merchlinsky M, Sisler J R,Moss B Marker rescue of the host range restriction defects of modifiedvaccinia virus Ankara. Virology 1998 251:334-42

[0298] Wyatt L S, Shors S T, Murphy B R, Moss B Development of areplication-deficient recombinant vaccinia virus vaccine effectiveagainst parainfluenza virus 3 infection in an animal model. Vaccine 1996Oct; 14(15):1451-8

1 64 1 381 RNA Equine infectious anemia virus 1 augauaccgg gcacucagauucugcggucu gagucccuuc ucugcugggc ugaaaaggcc 60 uuuguauaaa uauaauucucuacucagucc cugucucuag uuugucuguu cgagauccua 120 caguuggcgc ccgaacagggaccugagggg gcgcagaccc uaccuguuga accuggcuga 180 ucguaggauc cccgggacagcagaggagaa cuuacagaag ucuucuggag guguuccugg 240 ggagaacaca ggaggacagguaagauggga gacccuuuga cauggagcaa ggcgcucaag 300 aaguuaagaa ggugacgguacaagggucuc aguuaacucu gguaacugua auugggcgcu 360 aagucuaggu agacuuauuu c381 2 41 DNA Artificial Sequence misc_feature (1)..(41) sequence showingpart of split polyA signal 2 tcgctgcagc ggaataaagg gcaggtaagt atcaaggttac 41 3 60 DNA Artificial Sequence, primer misc_feature (1)..(60)sequence showing the part of split polyA signal 3 tcgctgcagc ggacacacaaaaaaccaaca cacagaactg ggaagtggac acctgtggag 60 4 63 DNA ArtificialSequence misc_feature (1)..(63) sequence showing both the parts of polyAsignal 4 aataaagggc aggtaagctc cacaggtgtc cactccagtt ctgtgtgttggttttttgtg 60 tgt 63 5 50 DNA Artificial Sequence polyA_signal (1)..(50)sequence of the polyA signal 5 aataaagggc aggtgtccac tccagttctgtgtgttggtt ttttgtgtgt 50 6 33 DNA Artificial Sequence, primermisc_feature (1)..(33) primer 6 tcgatagatc tgagtccgtt acataactta cgg 337 57 DNA Artificial Sequence,primer misc_feature (1)..(57) primer 7gatctcgaac agacaaacta gagacaggga ctgcaaacag caagaggctt tattggg 57 8 30DNA Artificial Sequence,primer misc_feature (1)..(30) primer 8gtccctgtct ctagtttgtc tgttcgagat 30 9 27 DNA Artificial Sequence,primermisc_feature (1)..(27) primer 9 ggggatccac tagttctaga gatattc 27 10 27DNA Artificial Sequence,primer misc_feature (1)..(27) primer 10ccttagacct ggagattcga agcgaag 27 11 53 DNA Artificial Sequence,primermisc_feature (1)..(53) primer 11 ccaaacctac aggtggggtc tttcatttacaaggttatga gagcatcagc aac 53 12 27 DNA Artificial Sequence,primermisc_feature (1)..(27) primer 12 aatgaaagac cccacctgta ggtttgg 27 13 41DNA Artificial Sequence,primer misc_feature (1)..(41) primer 13gtagagtgcc caattgccag tatacactcc gctatcgcta c 41 14 11299 DNA ArtificialSequence misc_feature (1)..(11299) plasmid 14 ctaaattgta agcgttaatattttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccgaaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttccagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaaccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggtcgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgacggggaaagcc aacctggctt atcgaaatta atacgactca 360 ctatagggag accggcagatctgagtccgt tacataactt acggtaaatg gcccgcctgg 420 ctgaccgccc aacgacccccgcccattgac gtcaataatg acgtatgttc ccatagtaac 480 gccaataggg actttccattgacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 540 ggcagtacat caagtgtatcatatgccaag tacgccccct attgacgtca atgacggtaa 600 atggcccgcc tggcattatgcccagtacat gaccttatgg gactttccta cttggcagta 660 catctacgta ttagtcatcgctattaccat ggtgatgcgg ttttggcagt acatcaatgg 720 gcgtggatag cggtttgactcacggggatt tccaagtctc caccccattg acgtcaatgg 780 gagtttgttt tggcaccaaaatcaacggga ctttccaaaa tgtcgtaaca actccgcccc 840 attgacgcaa atgggcggtaggcgtgtacg gtgggaggtc tatataagca gagctcgttt 900 agtgaaccgc gccagtcttccgatagactg cgtcgcccgg gtacccgtat tcccaataaa 960 gcctcttgct gtttgcatccgaatcgtggt ctcgctgttc cttgggaggg tctcctctga 1020 gtgattgact acccacgacgggggtctttc atttctctag tttgtctgtt cgagatccta 1080 cagttggcgc ccgaacagggacctgagagg ggcgcagacc ctacctgttg aacctggctg 1140 atcgtaggat ccccgggacagcagaggaga acttacagaa gtcttctgga ggtgttcctg 1200 gccagaacac aggaggacaggtaagatggg agaccctttg acatggagca aggcgctcaa 1260 gaagttagag aaggtgacggtacaagggtc tcagaaatta actactggta actgtaattg 1320 ggcgctaagt ctagtagacttatttcatga taccaacttt gtaaaagaaa aggactggca 1380 gctgagggat gtcattccattgctggaaga tgtaactcag acgctgtcag gacaagaaag 1440 agaggccttt gaaagaacatggtgggcaat ttctgctgta aagatgggcc tccagattaa 1500 taatgtagta gatggaaaggcatcattcca gctcctaaga gcgaaatatg aaaagaagac 1560 tgctaataaa aagcagtctgagccctctga agaatatctc tagagtgtga ttttaagggc 1620 gaattctgca ggagtggggaggcacgatgg ccgctttggt cgaggcggat ccggccatta 1680 gccatattat tcattggttatatagcataa atcaatattg gctattggcc attgcatacg 1740 ttgtatccat atcataatatgtacatttat attggctcat gtccaacatt accgccatgt 1800 tgacattgat tattgactagttattaatag taatcaatta cggggtcatt agttcatagc 1860 ccatatatgg agttccgcgttacataactt acggtaaatg gcccgcctgg ctgaccgccc 1920 aacgaccccc gcccattgacgtcaataatg acgtatgttc ccatagtaac gccaataggg 1980 actttccatt gacgtcaatgggtggagtat ttacggtaaa ctgcccactt ggcagtacat 2040 caagtgtatc atatgccaagtacgccccct attgacgtca atgacggtaa atggcccgcc 2100 tggcattatg cccagtacatgaccttatgg gactttccta cttggcagta catctacgta 2160 ttagtcatcg ctattaccatggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 2220 cggtttgact cacggggatttccaagtctc caccccattg acgtcaatgg gagtttgttt 2280 tggcaccaaa atcaacgggactttccaaaa tgtcgtaaca actccgcccc attgacgcaa 2340 atgggcggta ggcatgtacggtgggaggtc tatataagca gagctcgttt agtgaaccgt 2400 cagatcgcct ggagacgccatccacgctgt tttgacctcc atagaagaca ccgggaccga 2460 tccagcctcc gcggccccaagcttcagctg ctcgaggatc tgcggatccg gggaattccc 2520 cagtctcagg atccaccatgggggatcccg tcgttttaca acgtcgtgac tgggaaaacc 2580 ctggcgttac ccaacttaatcgccttgcag cacatccccc tttcgccagc tggcgtaata 2640 gcgaagaggc ccgcaccgatcgcccttccc aacagttgcg cagcctgaat ggcgaatggc 2700 gctttgcctg gtttccggcaccagaagcgg tgccggaaag ctggctggag tgcgatcttc 2760 ctgaggccga tactgtcgtcgtcccctcaa actggcagat gcacggttac gatgcgccca 2820 tctacaccaa cgtaacctatcccattacgg tcaatccgcc gtttgttccc acggagaatc 2880 cgacgggttg ttactcgctcacatttaatg ttgatgaaag ctggctacag gaaggccaga 2940 cgcgaattat ttttgatggcgttaactcgg cgtttcatct gtggtgcaac gggcgctggg 3000 tcggttacgg ccaggacagtcgtttgccgt ctgaatttga cctgagcgca tttttacgcg 3060 ccggagaaaa ccgcctcgcggtgatggtgc tgcgttggag tgacggcagt tatctggaag 3120 atcaggatat gtggcggatgagcggcattt tccgtgacgt ctcgttgctg cataaaccga 3180 ctacacaaat cagcgatttccatgttgcca ctcgctttaa tgatgatttc agccgcgctg 3240 tactggaggc tgaagttcagatgtgcggcg agttgcgtga ctacctacgg gtaacagttt 3300 ctttatggca gggtgaaacgcaggtcgcca gcggcaccgc gcctttcggc ggtgaaatta 3360 tcgatgagcg tggtggttatgccgatcgcg tcacactacg tctgaacgtc gaaaacccga 3420 aactgtggag cgccgaaatcccgaatctct atcgtgcggt ggttgaactg cacaccgccg 3480 acggcacgct gattgaagcagaagcctgcg atgtcggttt ccgcgaggtg cggattgaaa 3540 atggtctgct gctgctgaacggcaagccgt tgctgattcg aggcgttaac cgtcacgagc 3600 atcatcctct gcatggtcaggtcatggatg agcagacgat ggtgcaggat atcctgctga 3660 tgaagcagaa caactttaacgccgtgcgct gttcgcatta tccgaaccat ccgctgtggt 3720 acacgctgtg cgaccgctacggcctgtatg tggtggatga agccaatatt gaaacccacg 3780 gcatggtgcc aatgaatcgtctgaccgatg atccgcgctg gctaccggcg atgagcgaac 3840 gcgtaacgcg aatggtgcagcgcgatcgta atcacccgag tgtgatcatc tggtcgctgg 3900 ggaatgaatc aggccacggcgctaatcacg acgcgctgta tcgctggatc aaatctgtcg 3960 atccttcccg cccggtgcagtatgaaggcg gcggagccga caccacggcc accgatatta 4020 tttgcccgat gtacgcgcgcgtggatgaag accagccctt cccggctgtg ccgaaatggt 4080 ccatcaaaaa atggctttcgctacctggag agacgcgccc gctgatcctt tgcgaatacg 4140 cccacgcgat gggtaacagtcttggcggtt tcgctaaata ctggcaggcg tttcgtcagt 4200 atccccgttt acagggcggcttcgtctggg actgggtgga tcagtcgctg attaaatatg 4260 atgaaaacgg caacccgtggtcggcttacg gcggtgattt tggcgatacg ccgaacgatc 4320 gccagttctg tatgaacggtctggtctttg ccgaccgcac gccgcatcca gcgctgacgg 4380 aagcaaaaca ccagcagcagtttttccagt tccgtttatc cgggcaaacc atcgaagtga 4440 ccagcgaata cctgttccgtcatagcgata acgagctcct gcactggatg gtggcgctgg 4500 atggtaagcc gctggcaagcggtgaagtgc ctctggatgt cgctccacaa ggtaaacagt 4560 tgattgaact gcctgaactaccgcagccgg agagcgccgg gcaactctgg ctcacagtac 4620 gcgtagtgca accgaacgcgaccgcatggt cagaagccgg gcacatcagc gcctggcagc 4680 agtggcgtct ggcggaaaacctcagtgtga cgctccccgc cgcgtcccac gccatcccgc 4740 atctgaccac cagcgaaatggatttttgca tcgagctggg taataagcgt tggcaattta 4800 accgccagtc aggctttctttcacagatgt ggattggcga taaaaaacaa ctgctgacgc 4860 cgctgcgcga tcagttcacccgtgcaccgc tggataacga cattggcgta agtgaagcga 4920 cccgcattga ccctaacgcctgggtcgaac gctggaaggc ggcgggccat taccaggccg 4980 aagcagcgtt gttgcagtgcacggcagata cacttgctga tgcggtgctg attacgaccg 5040 ctcacgcgtg gcagcatcaggggaaaacct tatttatcag ccggaaaacc taccggattg 5100 atggtagtgg tcaaatggcgattaccgttg atgttgaagt ggcgagcgat acaccgcatc 5160 cggcgcggat tggcctgaactgccagctgg cgcaggtagc agagcgggta aactggctcg 5220 gattagggcc gcaagaaaactatcccgacc gccttactgc cgcctgtttt gaccgctggg 5280 atctgccatt gtcagacatgtataccccgt acgtcttccc gagcgaaaac ggtctgcgct 5340 gcgggacgcg cgaattgaattatggcccac accagtggcg cggcgacttc cagttcaaca 5400 tcagccgcta cagtcaacagcaactgatgg aaaccagcca tcgccatctg ctgcacgcgg 5460 aagaaggcac atggctgaatatcgacggtt tccatatggg gattggtggc gacgactcct 5520 ggagcccgtc agtatcggcggaattccagc tgagcgccgg tcgctaccat taccagttgg 5580 tctggtgtca aaaataataataaccgggca ggggggatcc gcagatccgg ctgtggaatg 5640 tgtgtcagtt agggtgtggaaagtccccag gctccccagc aggcagaagt atgcaaagca 5700 tgcctgcagg aattcgatatcaagcttatc gataccgtcg acctcgaggg ggggcccggt 5760 acccagcttt tgttccctttagtgagggtt aattgcgcgg gaagtattta tcactaatca 5820 agcacaagta atacatgagaaacttttact acagcaagca caatcctcca aaaaattttg 5880 tttttacaaa atccctggtgaacatgattg gaagggacct actagggtgc tgtggaaggg 5940 tgatggtgca gtagtagttaatgatgaagg aaagggaata attgctgtac cattaaccag 6000 gactaagtta ctaataaaaccaaattgagt attgttgcag gaagcaagac ccaactacca 6060 ttgtcagctg tgtttcctgaggtctctagg aattgattac ctcgatgctt cattaaggaa 6120 gaagaataaa caaagactgaaggcaatcca acaaggaaga caacctcaat atttgttata 6180 aggtttgata tatgggagtatttggtaaag gggtaacatg gtcagcatcg cattctatgg 6240 gggaatccca gggggaatctcaacccctat tacccaacag tcagaaaaat ctaagtgtga 6300 ggagaacaca atgtttcaaccttattgtta taataatgac agtaagaaca gcatggcaga 6360 atcgaaggaa gcaagagaccaagaaatgaa cctgaaagaa gaatctaaag aagaaaaaag 6420 aagaaatgac tggtggaaaataggtatgtt tctgttatgc ttagcaggaa ctactggagg 6480 aatactttgg tggtatgaaggactcccaca gcaacattat atagggttgg tggcgatagg 6540 gggaagatta aacggatctggccaatcaaa tgctatagaa tgctggggtt ccttcccggg 6600 gtgtagacca tttcaaaattacttcagtta tgagaccaat agaagcatgc atatggataa 6660 taatactgct acattattagaagctttaac caatataact gctctataaa taacaaaaca 6720 gaattagaaa catggaagttagtaaagact tctggcataa ctcctttacc tatttcttct 6780 gaagctaaca ctggactaattagacataag agagattttg gtataagtgc aatagtggca 6840 gctattgtag ccgctactgctattgctgct agcgctacta tgtcttatgt tgctctaact 6900 gaggttaaca aaataatggaagtacaaaat catacttttg aggtagaaaa tagtactcta 6960 aatggtatgg atttaatagaacgacaaata aagatattat atgctatgat tcttcaaaca 7020 catgcagatg ttcaactgttaaaggaaaga caacaggtag aggagacatt taatttaatt 7080 ggatgtatag aaagaacacatgtattttgt catactggtc atccctggaa tatgtcatgg 7140 ggacatttaa atgagtcaacacaatgggat gactgggtaa gcaaaatgga agatttaaat 7200 caagagatac taactacacttcatggagcc aggaacaatt tggcacaatc catgataaca 7260 ttcaatacac cagatagtatagctcaattt ggaaaagacc tttggagtca tattggaaat 7320 tggattcctg gattgggagcttccattata aaatatatag tgatgttttt gcttatttat 7380 ttgttactaa cctcttcgcctaagatcctc agggccctct ggaaggtgac cagtggtgca 7440 gggtcctccg gcagtcgttacctgaagaaa aaattccatc acaaacatgc atcgcgagaa 7500 gacacctggg accaggcccaacacaacata cacctagcag gcgtgaccgg tggatcaggg 7560 gacaaatact acaagcagaagtactccagg aacgactgga atggagaatc agaggagtac 7620 aacaggcggc caaagagctgggtgaagtca atcgaggcat ttggagagag ctatatttcc 7680 gagaagacca aaggggagatttctcagcct ggggcggcta tcaacgagca caagaacggc 7740 tctgggggga acaatcctcaccaagggtcc ttagacctgg agattcgaag cgaaggagga 7800 aacatttatg actgttgcattaaagcccaa gaaggaactc tcgctatccc ttgctgtgga 7860 tttcccttat ggctattttggggactagta attatagtag gacgcatagc aggctatgga 7920 ttacgtggac tcgctgttataataaggatt tgtattagag gcttaaattt gatatttgaa 7980 ataatcagaa aaatgcttgattatattgga agagctttaa atcctggcac atctcatgta 8040 tcaatgcctc agtatgtttagaaaaacaag gggggaactg tggggttttt atgaggggtt 8100 ttataaatga ttataagagtaaaaagaaag ttgctgatgc tctcataacc ttgtaaatga 8160 aagaccccac ctgtaggtttggcaagctag cttaagtaac gccattttgc aaggcatgga 8220 aaaatacata actgagaatagagaagttca gatcaaggtc aggaacagat ggaacagctg 8280 aatatgggcc aaacaggatatctgtggtaa gcagttcctg ccccggctca gggccaagaa 8340 cagatggaac agctgaatatgggccaaaca ggatatctgt ggtaagcagt tcctgccccg 8400 gctcagggcc aagaacagatggtccccaga tgcggtccag ccctcagcag tttctagaga 8460 accatcagat gtttccagggtgccccaagg acctgaaatg accctgtgcc ttatttgaac 8520 taaccaatca gttcgcttctcgcttctgtt cgcgcgcttc tgctccccga gctcaataaa 8580 agagcccaca acccctcactcggggcgcca gtcctccgat tgactgagtc gcccgggtac 8640 ccgtgtatcc aataaaccctcttgcagttg catccgactt gtggtctcgc tgttccttgg 8700 gagggtctcc tctgagtgattgactacccg tcagcggggg tctttcattt gggggctcgt 8760 ccgggatcgg gagacccctgcccagggacc accgacccac caccgggagg taagctggct 8820 gcctcgcgcg tttcggtgatgacggtgaaa acctctgaca catgcagctc ccggagacgg 8880 tcacagcttg tctgtaagcggatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 8940 gtgttggcgg gtgtcggggcgcagccatga cccagtcacg tagcgatagc ggagtgtata 9000 ctggcaattg ggcactcagattctgcggtc tgagtccctt ctctgctggg ctgaaaaggc 9060 ctttgtaata aatataattctctactcagt ccctgtctct agtttgtctg ttcgagatcc 9120 tacagagctc atgccttggcgtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 9180 ccgctcacaa ttccacacaacatacgagcc ggaagcataa agtgtaaagc ctggggtgcc 9240 taatgagtga gctaactcacattaattgcg ttgcgctcac tgcccgcttt ccagtcggga 9300 aacctgtcgt gccagctgcattaatgaatc ggccaacgcg cggggagagg cggtttgcgt 9360 attgggcgct cttccgcttcctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 9420 cgagcggtat cagctcactcaaaggcggta atacggttat ccacagaatc aggggataac 9480 gcaggaaaga acatgtgagcaaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 9540 ttgctggcgt ttttccataggctccgcccc cctgacgagc atcacaaaaa tcgacgctca 9600 agtcagaggt ggcgaaacccgacaggacta taaagatacc aggcgtttcc ccctggaagc 9660 tccctcgtgc gctctcctgttccgaccctg ccgcttaccg gatacctgtc cgcctttctc 9720 ccttcgggaa gcgtggcgctttctcatagc tcacgctgta ggtatctcag ttcggtgtag 9780 gtcgttcgct ccaagctgggctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 9840 ttatccggta actatcgtcttgagtccaac ccggtaagac acgacttatc gccactggca 9900 gcagccactg gtaacaggattagcagagcg aggtatgtag gcggtgctac agagttcttg 9960 aagtggtggc ctaactacggctacactaga aggacagtat ttggtatctg cgctctgctg 10020 aagccagtta ccttcggaaaaagagttggt agctcttgat ccggcaaaca aaccaccgct 10080 ggtagcggtg gtttttttgtttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 10140 gaagatcctt tgatcttttctacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 10200 gggattttgg tcatgagattatcaaaaagg atcttcacct agatcctttt aaattaaaaa 10260 tgaagtttta aatcaatctaaagtatatat gagtaaactt ggtctgacag ttaccaatgc 10320 ttaatcagtg aggcacctatctcagcgatc tgtctatttc gttcatccat agttgcctga 10380 ctccccgtcg tgtagataactacgatacgg gagggcttac catctggccc cagtgctgca 10440 atgataccgc gagacccacgctcaccggct ccagatttat cagcaataaa ccagccagcc 10500 ggaagggccg agcgcagaagtggtcctgca actttatccg cctccatcca gtctattaat 10560 tgttgccggg aagctagagtaagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 10620 attgctacag gcatcgtggtgtcacgctcg tcgtttggta tggcttcatt cagctccggt 10680 tcccaacgat caaggcgagttacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 10740 ttcggtcctc cgatcgttgtcagaagtaag ttggccgcag tgttatcact catggttatg 10800 gcagcactgc ataattctcttactgtcatg ccatccgtaa gatgcttttc tgtgactggt 10860 gagtactcaa ccaagtcattctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 10920 gcgtcaatac gggataataccgcgccacat agcagaactt taaaagtgct catcattgga 10980 aaacgttctt cggggcgaaaactctcaagg atcttaccgc tgttgagatc cagttcgatg 11040 taacccactc gtgcacccaactgatcttca gcatctttta ctttcaccag cgtttctggg 11100 tgagcaaaaa caggaaggcaaaatgccgca aaaaagggaa taagggcgac acggaaatgt 11160 tgaatactca tactcttcctttttcaatat tattgaagca tttatcaggg ttattgtctc 11220 atgagcggat acatatttgaatgtatttag aaaaataaac aaataggggt tccgcgcaca 11280 tttccccgaa aagtgccac11299 15 66 DNA Artificial Sequence,primer misc_feature (1)..(66) primer15 atcgaagctt aattaaaagt agaaaatata ttctaattta ttgggcactc agttctgcgg 60tctgag 66 16 35 DNA Artificial Sequence,primer misc_feature (1)..(35)primer 16 tcagctgcag ttcgggcgcc aactgtagga tctcg 35 17 33 DNA ArtificialSequence,primer misc_feature (1)..(33) primer 17 actgctgcag agattcgaagcgaaggagga aac 33 18 31 DNA Artificial Sequence,primer misc_feature(1)..(31) primer 18 tgtggggttt ccatgagggg ttttataaat g 31 19 30 DNAArtificial Sequence,primer misc_feature (1)..(30) primer 19 ccctcatggaaaccccacgt tccccccttg 30 20 33 DNA Artificial Sequence,primermisc_feature (1)..(33) primer 20 ctgaagatct gaatctgagt gcccaattgt cag 3321 23 DNA Artificial Sequence,primer misc_feature (1)..(23) primer 21ctgacaattg ggcactcaga ttc 23 22 44 DNA Artificial Sequence,primermisc_feature (1)..(44) primer 22 catgagatct taaaaaaaaa tgatgagagaattatattta ttac 44 23 21 DNA Equine infectious anemia virus misc_feature(1)..(21) 23 gggcactcag attctgcggt c 21 24 77 DNA Equine infectiousanemia virus 24 cuagugauuc ugagugcccc ugaugagcgg ccgaaaggcc gcgaaaccugcguacgacac 60 gcaggucggg cactcag 77 25 50 DNA Artificial Sequencepromoter (13)..(29) T7 promoter 25 atcgttaatt aataatacga ctcactatagggcactcaga ttctgcggtc 50 26 82 DNA Artificial Sequence terminator(11)..(59) T7 termination sequence 26 catgagatct caaaaaaccc ctcaagacccgtttagaggc cccaaggggt tatgctagtg 60 atgagagaat tatatttatt ac 82 27 7252DNA Artificial Sequence, plasmid misc_feature (1)..(7252) plasmid vector27 agcttttgcg atcaataaat ggatcacaac cagtatctct taacgatgtt cttcgcagat 60gatgattcat tttttaagta tttggctagt caagatgatg aaatcttcat tatctgatat 120attgcaaatc actcaatatc tagactttct gttattatta ttgatccaat caaaaaataa 180attagaagcc gtgggtcatt gttatgaatc tctttcagag gaatacagac aattgacaaa 240attcacagac tttcaagatt ttaaaaaact gtttaacaag gtccctattg ttacagatgg 300aagggtcaaa cttaataaag gatatttgtt cgactttgtg attagtttga tgcgattcaa 360aaaagaatcc tctctagcta ccaccgcaat agatcctgtt agatacatag atcctcgtcg 420caatatcgca ttttctaacg tgatggatat attaaagtcg aataaagtga acaataatta 480attctttatt gtcatcatga acggcggaca tattcagttg ataatcggcc ccatgttttc 540aggtaaaagt acagaattaa ttagacgagt tagacgttat caaatagctc aatataaatg 600cgtgactata aaatattcta acgataatag atacggaacg ggactatgga cgcatgataa 660gaataatttt gaagcattgg aagcaactaa actatgtgat ctcttggaat caattacaga 720tttctccgtg ataggtatcg atgaaggaca gttctttcca gacattgttg aattagatcg 780ataaaaatta attaattacc cgggtaccag gcctagatct gtcgacttcg agcttattta 840tattccaaaa aaaaaaaata aaatttcaat ttttaagctt tcactaattc caaacccacc 900cgctttttat agtaagtttt tcacccataa ataataaata caataattaa tttctcgtaa 960aagtagaaaa tatattctaa tttattgcac ggtaaggaag tagatcataa ctcgagcatg 1020ggagatcccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 1080cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 1140cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca 1200ccagaagcgg tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc 1260gtcccctcaa actggcagat gcacggttac gatgcgccca tctacaccaa cgtaacctat 1320cccattacgg tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc 1380acatttaatg ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc 1440gttaactcgg cgtttcatct gtggtgcaac gggcgctggg tcggttacgg ccaggacagt 1500cgtttgccgt ctgaatttga cctgagcgca tttttacgcg ccggagaaaa ccgcctcgcg 1560gtgatggtgc tgcgttggag tgacggcagt tatctggaag atcaggatat gtggcggatg 1620agcggcattt tccgtgacgt ctcgttgctg cataaaccga ctacacaaat cagcgatttc 1680catgttgcca ctcgctttaa tgatgatttc agccgcgctg tactggaggc tgaagttcag 1740atgtgcggcg agttgcgtga ctacctacgg gtaacagttt ctttatggca gggtgaaacg 1800caggtcgcca gcggcaccgc gcctttcggc ggtgaaatta tcgatgagcg tggtggttat 1860gccgatcgcg tcacactacg tctcaacgtc gaaaacccga aactgtggag cgccgaaatc 1920ccgaatctct atcgtgcggt ggttgaactg cacaccgccg acggcacgct gattgaagca 1980gaagcctgcg atgtcggttt ccgcgaggtg cggattgaaa atggtctgct gctgctgaac 2040ggcaagccgt tgctgattcg aggcgttaac cgtcacgagc atcatcctct gcatggtcag 2100gtcatggatg agcagacgat ggtgcaggat atcctgctga tgaagcagaa caactttaac 2160gccgtgcgct gttcgcatta tccgaaccat ccgctgtggt acacgctgtg cgaccgctac 2220ggcctgtatg tggtggatga agccaatatt gaaacccacg gcatggtgcc aatgaatcgt 2280ctgaccgatg atccgcgctg gctaccggcg atgagcgaac gcgtaacgcg aatggtgcag 2340cgcgatcgta atcacccgag tgtgatcatc tggtcgctgg ggaatgaatc aggccacggc 2400gctaatcacg acgcgctgta tcgctggatc aaatctgtcg atccttcccg cccggtgcag 2460tatgaaggcg gcggagccga caccacggcc accgatatta tttgcccgat gtacgcgcgc 2520gtggatgaag accagccctt cccggctgtg ccgaaatggt ccatcaaaaa atggctttcg 2580ctacctggag agacgcgccc gctgatcctt tgcgaatacg cccacgcgat gggtaacagt 2640cttggcggtt tcgctaaata ctggcaggcg tttcgtcagt atccccgttt acagggcggc 2700ttcgtctggg actgggtgga tcagtcgctg attaaatatg atgaaaacgg caacccgtgg 2760tcggcttacg gcggtgattt tggcgatacg ccgaacgatc gccagttctg tatgaacggt 2820ctggtctttg ccgaccgcac gccgcatcca gcgctgacgg aagcaaaaca ccagcagcag 2880tttttccagt tccgtttatc cgggcaaacc atcgaagtga ccagcgaata cctgttccgt 2940catagcgata acgagctcct gcactggatg gtggcgctgg atggtaagcc gctggcaagc 3000ggtgaagtgc ctctggatgt cgctccacaa ggtaaacagt tgattgaact gcctgaacta 3060ccgcagccgg agagcgccgg gcaactctgg ctcacagtac gcgtagtgca accgaacgcg 3120accgcatggt cagaagccgg gcacatcagc gcctggcagc agtggcgtct ggcggaaaac 3180ctcagtgtga cgctccccgc cgcgtcccac gccatcccgc atctgaccac cagcgaaatg 3240gatttttgca tcgagctggg taataagcgt tggcaattta accgccagtc aggctttctt 3300tcacagatgt ggattggcga taaaaaacaa ctgctgacgc cgctgcgcga tcagttcacc 3360cgtgcaccgc tggataacga cattggcgta agtgaagcga cccgcattga ccctaacgcc 3420tgggtcgaac gctggaaggc ggcgggccat taccaggccg aagcagcgtt gttgcagtgc 3480acggcagata cacttgctga tgcggtgctg attacgaccg ctcacgcgtg gcagcatcag 3540gggaaaacct tatttatcag ccggaaaacc taccggattg atggtagtgg tcaaatggcg 3600attaccgttg atgttgaagt ggcgagcgat acaccgcatc cggcgcggat tggcctgaac 3660tgccagctgg cgcaggtagc agagcgggta aactggctcg gattagggcc gcaagaaaac 3720tatcccgacc gccttactgc cgcctgtttt gaccgctggg atctgccatt gtcagacatg 3780tataccccgt acgtcttccc gagcgaaaac ggtctgcgct gcgggacgcg cgaattgaat 3840tatggcccac accagtggcg cggcgacttc cagttcaaca tcagccgcta cagtcaacag 3900caactgatgg aaaccagcca tcgccatctg ctgcacgcgg aagaaggcac atggctgaat 3960atcgacggtt tccatatggg gattggtggc gacgactcct ggagcccgtc agtatcggcg 4020gaattcagct gagcgccggt cgctaccatt accagttggt ctggtgtcaa aaataataat 4080aaccgggcag gggggatcct tctgtgagcg tatggcaaac gaaggaaaaa tagttatagt 4140agccgcactc gatgggacat ttcaacgtaa accgtttaat aatattttga atcttattcc 4200attatctgaa atggtggtaa aactaactgc tgtgtgtatg aaatgcttta aggaggcttc 4260cttttctaaa cgattgggtg aggaaaccga gatagaaata ataggaggta atgatatgta 4320tcaatcggtg tgtagaaagt gttacatcga ctcataatat tatatttttt atctaaaaaa 4380ctaaaaataa acattgatta aattttaata taatacttaa aaatggatgt tgtgtcgtta 4440gataaaccgt ttatgtattt tgaggaaatt gataatgagt tagattacga accagaaagt 4500gcaaatgagg tcgcaaaaaa actgccgtat caaggacagt taaaactatt actaggagaa 4560ttattttttc ttagtaagtt acagcgacac ggtatattag atggtgccac cgtagtgtat 4620ataggatctg ctcccggtac acatatacgt tatttgagag atcatttcta taatttagga 4680gtgatcatca aatggatgct aattgacggc cgccatcatg atcctatttt aaatggattg 4740cgtgatgtga ctctagtgac tcggttcgtt gatgaggaat atctacgatc catcaaaaaa 4800caactgcatc cttctaagat tattttaatt tctgatgtga gatccaaacg aggaggaaat 4860gaacctagta cggcggattt actaagtaat tacgctctac aaaatgtcat gattagtatt 4920ttaaaccccg tggcgtctag tcttaaatgg agatgcccgt ttccagatca atggatcaag 4980gacttttata tcccacacgg taataaaatg ttacaacctt ttgctccttc atattcagct 5040gaaatgagat tattaagtat ttataccggt gagaacatga gactgactcg ggccgcgttg 5100ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 5160cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 5220ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 5280tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 5340gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 5400tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 5460gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 5520tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 5580ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 5640agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 5700gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 5760attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 5820agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 5880atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 5940cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 6000ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 6060agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 6120tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 6180gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 6240caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 6300ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 6360gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 6420tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 6480tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 6540cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 6600cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 6660gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 6720atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 6780agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 6840ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 6900aataggcgta tcacgaggcc ctttcgtctt cgaataaata cctgtgacgg aagatcactt 6960cgcagaataa ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg 7020gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag 7080atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat 7140ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca 7200ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc ag 7252 28 7387DNA Artificial Sequence, primer misc_feature (1)..(7387) plasmid vector28 cctcctgaaa aactggaatt taatacacca tttgtgttca tcatcagaca tgatattact 60ggatttatat tgtttatggg taaggtagaa tctccttaat atgggtacgg tgtaaggaat 120cattatttta tttatattga tgggtacgtg aaatctgaat tttcttaata aatattattt 180ttattaaatg tgtatatgtt gttttgcgat agccatgtat ctactaatca gatctattag 240agatattatt aattctggtg caatatgaca aaaattatac actaattagc gtctcgtttc 300agacatggat ctgtcacgaa ttaatacttg gaagtctaag cagctgaaaa gctttctctc 360tagcaaagat gcatttaagg cggatgtcca tggacatagt gccttgtatt atgcaatagc 420tgataataac gtgcgtctag tatgtacgtt gttgaacgct ggagcattga aaaatcttct 480agagaatgaa tttccattac atcaggcagc cacattggaa gataccaaaa tagtaaagat 540tttggctatt cagtggactg gatgattcga ggtacccgat cccccctgcc cggttattat 600tatttttgac accagaccaa ctggtaatgg tagcgaccgg cgctcagctg aattccgccg 660atactgacgg gctccaggag tcgtcgccac caatccccat atggaaaccg tcgatattca 720gccatgtgcc ttcttccgcg tgcagcagat ggcgatggct ggtttccatc agttgctgtt 780gactgtagcg gctgatgttg aactggaagt cgccgcgcca ctggtgtggg ccataattca 840attcgcgcgt cccgcagcgc agaccgtttt cgctcgggaa gacgtacggg gtatacatgt 900ctgacaatgg cagatcccag cggtcaaaac aggcggcagt aaggcggtcg ggatagtttt 960cttgcggccc taatccgagc cagtttaccc gctctgctac ctgcgccagc tggcagttca 1020ggccaatccg cgccggatgc ggtgtatcgc tcgccacttc aacatcaacg gtaatcgcca 1080tttgaccact accatcaatc cggtaggttt tccggctgat aaataaggtt ttcccctgat 1140gctgccacgc gtgagcggtc gtaatcagca ccgcatcagc aagtgtatct gccgtgcact 1200gcaacaacgc tgcttcggcc tggtaatggc ccgccgcctt ccagcgttcg acccaggcgt 1260tagggtcaat gcgggtcgct tcacttacgc caatgtcgtt atccagcggt gcacgggtga 1320actgatcgcg cagcggcgtc agcagttgtt ttttatcgcc aatccacatc tgtgaaagaa 1380agcctgactg gcggttaaat tgccaacgct tattacccag ctcgatgcaa aaatccattt 1440cgctggtggt cagatgcggg atggcgtggg acgcggcggg gagcgtcaca ctgaggtttt 1500ccgccagacg ccactgctgc caggcgctga tgtgcccggc ttctgaccat gcggtcgcgt 1560tcggttgcac tacgcgtact gtgagccaga gttgcccggc gctctccggc tgcggtagtt 1620caggcagttc aatcaactgt ttaccttgtg gagcgacatc cagaggcact tcaccgcttg 1680ccagcggctt accatccagc gccaccatcc agtgcaggag ctcgttatcg ctatgacgga 1740acaggtattc gctggtcact tcgatggttt gcccggataa acggaactgg aaaaactgct 1800gctggtgttt tgcttccgtc agcgctggat gcggcgtgcg gtcggcaaag accagaccgt 1860tcatacagaa ctggcgatcg ttcggcgtat cgccaaaatc accgccgtaa gccgaccacg 1920ggttgccgtt ttcatcatat ttaatcagcg actgatccac ccagtcccag acgaagccgc 1980cctgtaaacg gggatactga cgaaacgcct gccagtattt agcgaaaccg ccaagactgt 2040tacccatcgc gtgggcgtat tcgcaaagga tcagcgggcg cgtctctcca ggtagcgaaa 2100gccatttttt gatggaccat ttcggcacag ccgggaaggg ctggtcttca tccacgcgcg 2160cgtacatcgg gcaaataata tcggtggccg tggtgtcggc tccgccgcct tcatactgca 2220ccgggcggga aggatcgaca gatttgatcc agcgatacag cgcgtcgtga ttagcgccgt 2280ggcctgattc attccccagc gaccagatga tcacactcgg gtgattacga tcgcgctgca 2340ccattcgcgt tacgcgttcg ctcatcgccg gtagccagcg cggatcatcg gtcagacgat 2400tcattggcac catgccgtgg gtttcaatat tggcttcatc caccacatac aggccgtagc 2460ggtcgcacag cgtgtaccac agcggatggt tcggataatg cgaacagcgc acggcgttaa 2520agttgttctg cttcatcagc aggatatcct gcaccatcgt ctgctcatcc atgacctgac 2580catgcagagg atgatgctcg tgacggttaa cgcctcgaat cagcaacggc ttgccgttca 2640gcagcagcag accattttca atccgcacct cgcggaaacc gacatcgcag gcttctgctt 2700caatcagcgt gccgtcggcg gtgtgcagtt caaccaccgc acgatagaga ttcgggattt 2760cggcgctcca cagtttcggg ttttcgacgt tgagacgtag tgtgacgcga tcggcataac 2820caccacgctc atcgataatt tcaccgccga aaggcgcggt gccgctggcg acctgcgttt 2880caccctgcca taaagaaact gttacccgta ggtagtcacg caactcgccg cacatctgaa 2940cttcagcctc cagtacagcg cggctgaaat catcattaaa gcgagtggca acatggaaat 3000cgctgatttg tgtagtcggt ttatgcagca acgagacgtc acggaaaatg ccgctcatcc 3060gccacatatc ctgatcttcc agataactgc cgtcactcca acgcagcacc atcaccgcga 3120ggcggttttc tccggcgcgt aaaaatgcgc tcaggtcaaa ttcagacggc aaacgactgt 3180cctggccgta accgacccag cgcccgttgc accacagatg aaacgccgag ttaacgccat 3240caaaaataat tcgcgtctgg ccttcctgta gccagctttc atcaacatta aatgtgagcg 3300agtaacaacc cgtcggattc tccgtgggaa caaacggcgg attgaccgta atgggatagg 3360ttacgttggt gtagatgggc gcatcgtaac cgtgcatctg ccagtttgag gggacgacga 3420cagtatcggc ctcaggaaga tcgcactcca gccagctttc cggcaccgct tctggtgccg 3480gaaaccaggc aaagcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 3540tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 3600gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggga tctcccatgc 3660tcgagttatg atctacttcc ttaccgtgca ataaattaga atatattttc tacttttacg 3720agaaattaat tattgtattt attatttatg ggtgaaaaac ttactataaa aagcgggtgg 3780gtttggaatt agtgaaagct gggagatctg gcgcgcctgc agagaattcg tttaaacgga 3840tcccgagctt atttatattc caaaaaaaaa aaataaaatt tcaattttta agctggggat 3900cctctagagt cgacctgcag gcatgctcga gcggccgcca gtgtgatgga tatctgcaga 3960attcggcttg gggggctgca ggtggatgcg atcatgacgt cctctgcaat ggataacaat 4020gaacctaaag tactagaaat ggtatatgat gctacaattt tacccgaagg tagtagcatg 4080gattgtataa acagacacat caatatgtgt atacaacgca cctatagttc tagtataatt 4140gccatattgg atagattcct aatgatgaac aaggatgaac taaataatac acagtgtcat 4200ataattaaag aatttatgac atacgaacaa atggcgattg accattatgg agaatatgta 4260aacgctattc tatatcaaat tcgtaaaaga cctaatcaac atcacaccat taatctgttt 4320aaaaaaataa aaagaacccg gtatgacact tttaaagtgg atcccgtaga attcgtaaaa 4380aaagttatcg gatttgtatc tatcttgaac aaatataaac cggtttatag ttacgtcctg 4440tacgagaacg tcctgtacga tgagttcaaa tgtttcattg actacgtgga aactaagtat 4500ttctaaaatt aatgatgcat taatttttgt attgattctc aatcctaaaa actaaaatat 4560gaataagtat taaacatagc ggtgtactaa ttgatttaac ataaaaaata gttgttaact 4620aatcatgagg actctactta ttagatatat tctttggaga aatgacaacg atcaaaccgg 4680gcatgcaagc ttgtctccct atagtgagtc gtattagagc ttggcgtaat catggtcata 4740gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 4800cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 4860ctcactgccc gctttcgagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 4920acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 4980gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5040gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 5100ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc gataggctcc gcccccctga 5160cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5220ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 5280taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 5340ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 5400ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 5460aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 5520tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 5580agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 5640ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 5700tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 5760tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 5820cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 5880aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 5940atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 6000cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 6060tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 6120atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 6180taatagtttg cgcaacgttg ttggcattgc tacaggcatc gtggtgtcac gctcgtcgtt 6240tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 6300gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 6360cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 6420cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 6480gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 6540aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 6600accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 6660ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 6720gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 6780aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 6840taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 6900cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc 6960gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc 7020ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg 7080cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca 7140tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc 7200gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 7260ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 7320ccagtcacga cgttgtaaaa cgacggccag tgaattggat ttaggtgaca ctatagaata 7380cgaattc 7387 29 27 DNA Artificial Sequence, primer misc_feature(1)..(27) primer 29 gcatggacct gtggggtttt tatgagg 27 30 29 DNAArtificial Sequence, primer misc_feature (1)..(29) primer 30 gcatgagctctgtaggatct cgaacagac 29 31 33 DNA Artificial Sequence, primermisc_feature (1)..(33) primer 31 gactacgact agtgtatgtt tagaaaaaca agg 3332 32 DNA Artificial Sequence misc_feature (1)..(32) primer 32ctaggctact agtactgtag gatctcgaac ag 32 33 33 DNA Artificial Sequence,primer misc_feature (1)..(33) primer 33 gggctatatg agatcttgaa taataaaatgtgt 33 34 14 DNA Artificial Sequence, primer misc_feature (1)..(14)primer 34 tattaataac tagt 14 35 42 DNA Artificial Sequence, primermisc_feature (1)..(42) primer 35 gctacgcaga gctcgtttag tgaaccgggcactcagattc tg 42 36 36 DNA Artificial Sequence, primer misc_feature(1)..(36) primer 36 gctgagctct agagtccttt tcttttacaa agttgg 36 37 31 DNAArtificial Sequence, primer misc_feature (1)..(31) primer 37 gtcgctgaggtcgacaaggc aaagagaaga g 31 38 31 DNA Artificial Sequence, primermisc_feature (1)..(31) primer 38 gaccggtacc gtcgacaagg cacagcagtg g 3139 32 DNA Artificial Sequence, primer misc_feature (1)..(32) primer 39ttctgtcgac gaatcccagg gggaatctca ac 32 40 35 DNA Artificial Sequence,primer misc_feature (1)..(35) primer 40 gtcaccttcc agagggccct ggctaagcataacag 35 41 35 DNA Artificial Sequence, primer misc_feature (1)..(35)primer 41 ctgttatgct tagccagggc cctctggaag gtgac 35 42 28 DNA ArtificialSequence, primer misc_feature (1)..(28) primer 42 aattgctgac ccccaaaatagccataag 28 43 36 DNA Artificial Sequence, primer misc_feature (1)..(36)primer 43 ccatgcacgt ctgcagccag catggcagaa tcgaag 36 44 30 DNAArtificial Sequence, primer misc_feature (1)..(30) primer 44 cctgaggatctattttccac cagtcatttc 30 45 29 DNA Artificial Sequence, primermisc_feature (1)..(29) primer 45 gtggaaaata gatcctcagg gccctctgg 29 4634 DNA Artificial Sequence, primer misc_feature (1)..(34) primer 46gcagtgccgg atcctcataa atgtttcctc cttc 34 47 24 DNA Artificial Sequence,primer misc_feature (1)..(24) primer 47 gacaccatgg gaagtattta tcac 24 4835 DNA Artificial Sequence, primer misc_feature (1)..(35) primer 48cctgggattc atatcaaacc ttataacaaa tattg 35 49 22 DNA Artificial Sequence,primer misc_feature (1)..(22) primer 49 tcctgctaag cataacagaa ac 22 5029 DNA Artificial Sequence, primer misc_feature (1)..(29) primer 50ggtttgatat gaatcccagg gggaatctc 29 51 22 DNA Artificial Sequence, primermisc_feature (1)..(22) primer 51 accccgtacg tcttcccgag cg 22 52 39 DNAArtificial Sequence, primer misc_feature (1)..(39) primer 52 gttattaattaatggaggaa taattgaaga aggatatac 39 53 31 DNA Artificial Sequence, primermisc_feature (1)..(31) primer 53 tcttctgcag gtcctgatcc ttgcttagtg c 3154 41 DNA Artificial Sequence, primer misc_feature (1)..(41) primer 54gaccatgtta cccctttacc attaactccc taatatcaaa c 41 55 44 DNA ArtificialSequence, primer misc_feature (1)..(44) primer 55 gtaaaggggt aacatggtcagcatcgcatt ctacggggga atcc 44 56 38 DNA Artificial Sequence, primermisc_feature (1)..(38) primer 56 ccatgcacgt ctcgagccag catgggagaccctttgac 38 57 37 DNA Artificial Sequence, primer misc_feature (1)..(37)primer 57 cgagctagag gtcgactcaa tttggtttat tagtaac 37 58 32 DNAArtificial Sequence, primer misc_feature (1)..(32) primer 58 gcaatggaatgacatccctc agctgccagt cc 32 59 42 DNA Artificial Sequence, primermisc_feature (1)..(42) primer 59 gggatgtcat tccattgcca ccatgggaagtatttatcac ta 42 60 34 DNA Artificial Sequence, primer misc_feature(1)..(34) primer 60 gtcgagcacg cgtttgccta gcaacatgag ctag 34 61 34 DNAArtificial Sequence, primer misc_feature (1)..(34) primer 61 gtcgagccaattgttgccta gcaacatgag ctag 34 62 28 DNA Artificial Sequence misc_feature(1)..(28) P7.5E sequence 62 aaaagtagaa aatatattct aatttatt 28 63 19 DNAArtificial Sequence promoter (1)..(19) T7 promter 63 taatacgactcactatagg 19 64 48 DNA Artificial Sequence terminator (1)..(48) T7terminator 64 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttg 48

1. A retroviral vector derived from a non-primate lentivirus genomecomprising a deleted gag gene wherein the deletion in gag removes one ormore nucleotides downstream of nucleotide 350 of the gag codingsequence.
 2. A retroviral vector according to claim 1 wherein thedeletion extends from nucleotide 350 to at least the C-terminus of thegag-pol coding region.
 3. A retroviral vector according to claim 1 orclaim 2 wherein the deletion additionally removes nucleotide 300 of thegag coding region.
 4. A retroviral vector according to claim 1 whereinthe deletion retains the first 150 nucleotides of the gag coding region.5. A retroviral vector according to claim 1 wherein the deletion retainsthe first 109 nucleotides of the gag coding region.
 6. A retroviralvector according to claim 1 wherein the deletion retains only the first2 nucleotides of the gag coding region.
 7. A retroviral vector derivedfrom a non-primate lentivirus genome wherein one or more accessory genesare absent from the non-primate lentivirus genome.
 8. A retroviralvector according to claim 7 wherein the accessory genes are selectedfrom dUTPase, S2, env and tat.
 9. A retroviral vector derived from alentivirus genome wherein the non-primate lentivirus genome lacks thetat gene but includes the leader sequences between the end of the 5′ LTRand the ATG of gag.
 10. A retroviral vector according to any precedingclaim which comprises at least one component from an equininelentivirus.
 11. A retroviral vector according to claim 10 wherein theequinine lentivirus is EIAV.
 12. A retroviral vector according to claim11 wherein the retroviral vector is substantially derived from EIAV. 13.A retroviral vector production system for producing the retroviralvector of any preceding claim comprising a packaging cell comprising agag-pol gene from a non-primate lentivirus and an envelope gene.
 14. Aretroviral vector produced by the production system of claim
 13. 15. Ahybrid viral vector system comprising a primary viral vector derivedfrom a poxvirus and a second viral vector derived from a retrovirus. 16.A retroviral particle obtainable from the retroviral vector of any oneof claims 1 to 12 or claim 14 or
 15. 17. A cell transfected ortransduced with a retroviral vector according to any one of claims 1 to12 or claim 14 or 15 or a retroviral particle of claim
 16. 18. Aretroviral vector according to any one of claims 1 to 12 or claim 14 or15 or a retroviral particle of claim 16 or a cell according to claim 17for use in medicine.
 19. Use of a retroviral vector according to any oneof claims 1 to 12 or claim 14 or 15 or a retroviral particle of claim 16or a cell according to claim 17 for the manufacture of a pharmaceuticalcomposition to deliver an NOI to a target site in need of same.
 20. Amethod comprising transfecting or transducing a cell with a retroviralvector according to any one of claims 1 to 12 or claim 14 or 15 or aretroviral particle of claim 16 or by use of a cell according to claim17.
 21. A delivery system in the form of a retroviral vector accordingto any one of claims 1 to 12 or claim 14 or 15 or a retroviral particleof claim 16 or a cell according to claim
 17. 22. A delivery system for aretroviral vector according to any one of claims 1 to 12 or claim 14 or15 or a retroviral particle of claim 16 or a cell according to claim 17wherein the delivery system comprises a non-retroviral expressionvector, an adenovirus and/or a plasmid.
 23. A retroviral vectorsubstantially as hereinbefore described with reference to theaccompanying Figures.
 24. A retroviral vector production systemsubstantially as hereinbefore described with reference to theaccompanying Figures.
 25. A retroviral particle substantially ashereinbefore described with reference to the accompanying Figures.
 26. Acell transfected or transduced with a retroviral vector substantially ashereinbefore described with reference to the accompanying Figures. 27.Use of a retroviral vector substantially as hereinbefore described withreference to the accompanying Figures.
 28. A method comprisingtransfecting or transducing a cell with a retroviral vectorsubstantially as hereinbefore described with reference to theaccompanying Figures.
 29. A delivery system in the form of a retroviralvector substantially as hereinbefore described with reference to theaccompanying Figures.